Methods of identifying longevity modulators and therapeutic methods of use thereof

ABSTRACT

The present invention is based at least in part on the discovery of a role for the JNK signaling pathway in longevity. In particular, the present inventors have shown that overexpression of c-jun N-terminal kinase 1 (jnk-1) extends lifespan and that said extended lifespan is associated with DAF-16 phosphorylation by JNK-1 and the consequent DAF-16 localization to the nucleus. Accordingly, the present invention features methods of identifying modulators of longevity in assays featuring organisms and/or cells having a JNK signaling pathway and, optionally, an IR signaling pathway. Also featured is an in vitro method of identifying an agent capable of enhancing longevity featuring an assay composition having a JNK signaling pathway molecule and insulin signaling pathway molecule. Further featured are therapeutic methods for the use of JNK signaling pathway modulators to enhance longevity, to prevent or reduce obesity and to prevent or treat type II diabetes.

RELATED APPLICATIONS

This application is a continuation-in-part of co-pending U.S. UtilityApplication Ser. No. 10/746,910, entitled “Methods of IdentifyingLongevity Modulators and Therepeutic Methods of Use Thereof”, filed Dec.23, 2003, which claims the benefit of U.S. Provisional PatentApplication Ser. No: 60/436,324 entitled “Methods of IdentifyingLongevity Modulators and Therepeutic Methods of Use Thereof”, filed Dec.23, 2002. This application is also related to U.S. Provisional PatentApplication Ser. No: 60/660073, entitled “Methods of IdentifyingLongevity Modulators and Therepeutic Methods of Use Thereof”, filed Mar.7, 2005. The entire content of the referenced patent applications isincorporated herein by reference.

GOVERNMENT RIGHTS

This invention was made at least in part with government support undergrant no. DK32520-19 awarded by the National Institutes of Health. Thegovernment may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Aging in mammals can have a profound deleterious effect on brainfunction that manifests primarily in deficits of cognitive and motorfunction. Longevity genes are thus of obvious interest and importance,both for their life-extension potential and the possibility of theircontributing to the enhancement of the quality of life, particularlylater during the lifespan. However, very few of these genes have beenidentified and even less is understood about how these genes act toprevent aging and promote life extension.

Accordingly, there exists the need to discover genes whose function isassociated with life-extension. Such genes and their products would beuseful in the screening for anti-aging agents and would serve as keytargets in various anti-aging therapies. Indeed, an understanding of themechanisms underlying aging will ultimately provide us with the toolsnecessary to alleviate these deficits in the aged population and therebyprolong the independence of the elderly.

The roundworm C. elegans is a valuable invertebrate model system tostudy aging owing to its short, reproducible life span and itsamenability to genetic and molecular analysis. Further, as the entire C.elegans genome is sequenced, it is feasible to envisage a comprehensiveidentification of all the genes that affect aging in this organism. Inmolecular genetics, extended life span remains one of the bestindicators that an intervention in an aging process has been made. Thelife span of C. elegans is easily extendable by various genetic,transgenic, and pharmacologic means, and the isolation of long-livedvariants in C. elegans has begun to provide clear insights into theputative mechanisms and consequences of aging in the CNS.

The main pathway that regulates life span in C. elegans is aninsulin-like signaling pathway. Mutations in genes in this pathway canincrease, decrease, or have no effect on life span. Interestingly,several of these genes were first isolated based on their effects ondevelopment. The normal lifecycle of C. elegans follows development froman egg, through four larval stages, and a final molt into a fertile,adult hermaphrodite. When nutrition is low or population density ishigh, the worms can undergo an alternative developmental program to form“dauer” larvae (Cassada R. C. & Russell R. (1975) Dev. Biology46:326-342). The dauer larvae is a diapause stage that does not feed orreproduce, is stress resistant and is apparently non-aging, whereinworms can remain as dauer larvae for months (Klass M. R. & Hirsh D. I.(1976) Nature 260:523-525). When conditions improve, worms can re-enterthe life cycle and develop into a normal reproductive hermaphrodite. Thedauer formation genes (daf), or genes that determine the decision toprogress through development normally or undergo dauer formation, werefirst isolated on the basis that they either promote dauer arrest underplentiful growth conditions (dauer constitutive) or prevent dauerformation under crowded conditions (dauer defective) (Riddle D. L. etal. in C. elegans II, (1997) 739-768, Cold Spring Harbor LaboratoryPress). Several of these genes, including daf-2, age-1 and daf-16, weresubsequently identified as part of an insulin-like signaling pathway,supporting the idea that genes that affect entry into the dauer stagealso affect life span in C. elegans.

The insulin-like signaling pathway in C. elegans contains numerousgenes, many of which were isolated originally through their effects ondauer formation. Of the 37 insulin family members that have beenidentified in the C. elegans genome, only one insulin receptor-likegene, daf-2 (Pierce S. B. et al. (2001) Genes and Dev. 15:672-686;Gregoire F. M. et al. (1998). Biochem Biophys Res Com. 249:385-390) hasbeen clearly identified. DAF-2 highly resembles both the mammalianinsulin receptor and the related insulin growth factor-1 receptor(IGF1-R) (Kimura K. et al. (1997) Science 277:942-946). The ligand thatbinds to the DAF-2 receptor is not yet known. Activation of theinsulin-like receptor DAF-2 by the as yet unidentified ligand leads toactivation of PI-3 kinase, which in turn results in the generation ofphosphoinositide-3-phosphate (PIP₃). In mammalian systems, PIP₃ acts asan intracellular messenger to activate downstream kinases (Kido Y. etal. (2001) J of Clin End and Met 86: 972-979; Alessi, D. R. & Downes, C.P., (1998) Biochim Biophys Acta 1436: 151-164). In C. elegans, thecatalytic subunit, p110, of PI-3 kinase is encoded by the age-1 gene(Morris J. Z. et al. (1996) Nature 382:536-539). Decrease in functionmutations in either daf-2 or age-1 result in various phenotypesincluding constitutive dauer formation during development, fertilitydefects, resistance to stresses such as heat, oxidative damage and heavymetals, and extension of life span in adults (Lithgow G. J. et al.,(1994) J. Gerontol. 49:B270-276; Lithgow G. J. et al., (1995) PNAS USA92:7540-4; Murakami S. & Johnson T. E. A Genetics 143:1207-1218; HondaY. & Honda S., (1999) FASED J 13:1385-1393; Baryste D., (2001) FasEB J15:627-634; Friedman D. B. & Johnson T. E., (1988) Genetics 118:75-86;Klass M. R., (1983) Mech of Ageing and Dev. 22:279-286). Another gene inthe pathway, daf-18, encodes a homolog of the mammalian tumor suppressorPTEN phosphatase (Rouault J. P., (1999) Curr Biology 9:329-332; Ogg S. &Ruvkun G., (1998) Mol Cell 2:887-893; Mihaylova V. T. et al., (1999)PNAS USA 96:7427-7432; Gil E. B. et al. (1999) PNAS USA 96:2925-2930).DAF-18 functions to regulate the levels of PIP₃ by dephosphorylating theinositol ring in the third position (Maehama T. & Dixon J. E. (1998) JBiol Chem 273:13375-13378). Loss of function mutations in daf-18 resultin a decrease in life span and suppression of both daf-2 and age-1 dauerphenotypes (Rouault J. P. (1999) Curr Biol 9:329-332; Ogg S. & Ruvkun G.(1998) Mol Cell 2:887-893; Mihaylova V. T. (1999) PNAS USA 96:7427-7432;Gil E. B. et al. (1999) PNAS USA 96:2925-2930.)

Downstream of age-1 are the kinases PDK-1, AKT-1, and AKT-2. The PDK-1and AKT-1 kinases were identified in C. elegans as gain-of-functionsuppressors of the dauer-constitutive phenotype of age-1 mutants(Paradis S. & Ruvkun G. (1998) Genes Dev 12:2488-2498; Paradis S. (1999)Genes Dev 13:1438-1452). Similar to the phenotype observed for mutationsin daf-2 and age-1, a reduction of function mutation in PDK-1 increasesadult life span (Paradis S. (1999) Genes Dev 13:1438-1452). The finaloutput of the pathway is daf-16, which encodes a homolog of theHNF-3/forkhead family of transcription factors (Kimura K. et al. (1997)Science 277:942-946; Ogg S. et al. (1997) Nature 389:994-9; Lin K. etal. (1997) Science 278:1319-1322). Null mutations of daf-16 decreaselife span and completely suppress all phenotypes in double mutantcombinations with daf-2 or age-1. Thus life span extension by eitherdaf-2 or age-1 mutations requires a wild type daf-16 gene. Given thatDAF-1 and AGE-1 proteins act to suppress the activity of DAF-16, it isbelieved that the lack of signaling in daf-2 or age-1 mutants causesincreased activity of DAF-16, ultimately leading to the observedphenotypes. The final targets of DAF-16 in this pathway remain unknownbut are presumed to regulate metabolism and fat storage (Kimura K. etal. (1997) Science 277:942-946; Lithgow G. J. et al. (1995) PNAS USA92:7540-4).

In order to elucidate fully the mechanisms underlying aging, it will becritical to identify all pathways that play a role in its regulation.Importantly, genetic analysis of the DAF-2 insulin-like receptorstrongly indicates that other genes are involved in signaling downstreamof daf-2 (Ogg S. & Ruvkun G. (1998) Mol Cell 2:887-893). This is due tothe fact that a loss of function mutation in the daf-16 forkheadtranscription factor completely suppresses all of the phenotypes of aloss of function mutation in either daf-2 or age-1 (Kenyon, C. in C.elegans II (1997) 791-813, Cold Spring Harbor Press; Tissenbaum H. A. &Ruvkun G. (1998) Genetics 148:703-717)). However, loss of functionmutations in daf-18 as well as gain of function mutation in either akt-1and pdk-1 only suppress a subset of the phenotypes associated with thedaf-2 and age-1 loss of function mutations (Paradis S. & Ruvkun G.(1998) Genes Dev 12:2488-2498; Paradis S. et al. Genes Dev 13:1438-1452;Ogg S. & Ruvkun G. (1998) Mol Cell 2:887-893). These data indicate thatan additional pathway(s) is active downstream of daf-2 but upstream ofdaf-16. There exists, therefore, a clear need in the art for theelucidation of additional pathways involved in regulating aging.

Importantly, the influence of the insulin/IGF signaling pathway onlifespan has been conserved across large evolutionary distances. In thefruit fly Drosophila, reduced insulin/IGF signaling also mediateslife-span extensions (Clancy D. J. (2001) Science 292:104-106; Tatar M.& Yin C. (2001) Exp. Gerontol. 36:723-738). This conservation indicatesthat certain physiological processes effecting life span are veryancient and strongly suggests that information on the aging of simpleanimals is likely to be important for mammalian aging. The study ofdevelopment and longevity in C. elegans is thus expected to uncovercritical new targets for insulin regulators in higher organisms andpotential anti-aging targets for drug intervention in humans.

SUMMARY OF THE INVENTION

The present invention is based at least in part on the discovery of arole for the JNK signaling pathway in longevity. In particular, thepresent inventors have shown that modulation of the c-jun N-terminalkinase (JNK) signaling pathway in an organism, optionally in combinationwith modulation of the insulin receptor (IR) signaling pathway, canenhance longevity in an organism. Studies performed in the C. elegansmodel organism indicate that inhibition of JNK signaling, in particular,inhibition of JNK enzymatic activity, decreases lifespan and thatinhibition of other molecules in the JNK signaling pathway incombination with an inhibition of IR signaling can extend or decreaselifespan. Studies further indicate that enhancement of JNK signaling, inparticular, overexpression of jnk-1, extends lifespan and that saidextended lifespan is associated with DAF-16 phosphorylation by JNK-1 andthe consequent DAF-16 localization to the nucleus.

Accordingly, the present invention features methods of identifyingmodulators of longevity in assays featuring organisms and/or cellshaving either a functional or deregulated JNK signaling pathway and,optionally, a functional or deregulated IR signaling pathway.

In one embodiment, the invention provides a method for identifying anagent capable of enhancing longevity, involving contacting an organismhaving a deregulated c-jun N-terminal kinse (JNK) signaling pathway and,optionally, a deregulated IR signaling pathway, with a test agent,wherein the the test agent is identified based on its ability to alter aphenotype associated with the deregulated signaling pathway(s) ascompared to a suitable control.

In another embodiment, the invention provides a method for identifyingan agent capable of enhancing longevity, involving contacting anorganism having a deregulated c-jun N-terminal kinse (JNK) signalingpathway with a test agent, and identifying an agent based on its abilityto alter a downstream indicator of the c-jun N-terminal kinse (JNK)signaling pathway as compared to a suitable control.

Also featured are cell-based assays for the identification of an agentcapable of enhancing longevity. The invention features a methodinvolving contacting a cell having a JNK signaling pathway, optionallyin combination with an insulin receptor (IR) signaling pathway, with atest agent, and identifying the agent based on its ability to modulatethe signaling pathway(s) by detecting a signaling pathway indicator(s).

Also featured is an in vitro method of identifying an agent capable ofenhancing longevity. This method involves the steps of: (a) contacting afirst assay composition with a test compound, wherein the assaycomposition comprises a JNK signaling pathway molecule; (b) detectingactivity or expression of the JNK signaling pathway molecule; (c)contacting a second assay composition with the test compound, whereinthe assay composition comprises an insulin signaling pathway molecule;and (d) detecting activity or expression of the insulin signalingpathway molecule, wherein the agent is identified based on its abilityto modulate activity or expression of the JNK signaling pathway moleculeand insulin signaling pathway molecule.

Further featured are therapeutic methods for use of JNK signalingpathway modulators in order to enhance longevity in a subject, forexample, a human subject. In one embodiment, the invention features amethod for enhancing longevity in a subject, involving administering toa subject in need of enhanced longevity a pharmacologically effectivedose of an agent that modulates a JNK signaling pathway molecule,wherein the modulation of the JNK signaling pathway molecule in thesubject enhances longevity.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the insulin receptor (IR)signaling pathway.

FIG. 2 is a schematic representation of the c-jun N-terminal kinase(JNK) signaling pathway.

FIG. 3A-D is a graphical depiction of life span analysis of mutant C.elegans strains containing reduction of function mutations in jnk-1,unc-16, mek-1 and jkk-1 alone, and in combination with a reduction offunction daf-2 mutation.

FIG. 4 is a graphical depiction of life span analysis of mutant C.elegans strains containing reduction of function mutations jnk-1,unc-16, mek-1 and jkk-1 alone, and in combination with a reduction offunction age-1 mutation.

FIG. 5 is a graphical depiction of life span analysis of a double mutantC. elegans strain containing reduction of function mutations in daf-2and jnk-1, and a strain containing this same reduction of functionmutations in daf-2 and jnk-1 in combination with a null mutation indaf-16.

FIG. 6 is a graphical depiction of life span analysis of mutant C.elegans strains containing single reduction of function mutations injnk-1, unc-16, mek-1 and jkk-1.

FIG. 7A is a schematic representation of genomic DNA that was amplifiedand injected into C. elegans to construct a jnk-1 overexpressiontransgenic strain.

FIG. 7B shows the results of genomic PCR analysis from a single-wormdemonstrating the presence of extra copies of jnk-1 in a jnk-1overexpression transgenic strain.

FIG. 7C shows the results of RT-PCR analysis of a jnk-1 overexpressiontransgenic strain demonstrating a 10-fold overexpression of jnk-1 and abar graph quantitatively summarizing the data.

FIG. 8 is a graphical depiction of life span analysis of a jnk-1overexpression transgenic C. elegans strain.

FIG. 9 is a graphical depiction of life span analysis of a transgenic C.elegans strain in which jnk-1 is overexpressed or in which both jnk-1 isoverexpressed and daf-16 is knocked down by RNAi.

FIG. 10A is an image of GFP-tagged DAF-16 in a wild type C. elegansstrain.

FIG. 10B is an image of GFP-tagged DAF-16 in a daf-2 mutant C. elegansstrain.

FIG. 10C is an image of GFP-tagged DAF-16 in a jnk-1 overexpression C.elegans strain.

FIG. 11A is a graphical depiction of life span analysis of a mutant C.elegans strain containing a reduction of function mutation in jkk-1alone, and in combination with overexpression of jnk-1.

FIG. 11B is a graphical depiction of life span analysis of a mutant C.elegans strain containing a reduction of function mutation in mek-1alone, and in combination with overexpression of jnk-1.

FIG. 11C is a graphical depiction of life span analysis of a mutant C.elegans strain containing a reduction of function mutation in unc-16alone, and in combination with overexpression of jnk-1.

FIG. 12A is a graphical depiction of oxidative stress analysis of ajnk-1 overexpression C. elegans strain.

FIG. 12B is a graphical depiction of heat shock stress analysis of ajnk-1 overexpression C. elegans strain.

FIG. 13 is a model depicting the role of the JNK signaling pathway inlife span, wherein JNK signaling can regulate life span by modulatingnuclear translocation of DAF-16.

FIG. 14 is a bar graph showing that jnk-1 transgenic worms show anincrease in jnk-1 transcription level.

FIG. 15 is a graphical depiction of life span analysis of jnk-1overexpression lines lpIn1 and lpn2.

FIGS. 16A-C are graphical depictions of life span analysis showing thatjnk-1 regulates life spanin parallel to the insulin-like pathway butboth converge onto daf-16. (A) Life span analysis of jnk-1overexpression strain (lpIn1) on daf-16 RNAi. All life span data ispresented as mean life span±standard error (total number of animalsscored). N2 on control RNAi: 17.6±0.5 (125), N2 on daf-16 RNAi: 14.6±0.3(145), lpIn1 on control RNAi: 19.1±0.6 (147), lpIn1 on daf-16 RNAi:14.5±0.3 (136). (B) Life span analysis of daf-2(e1370); lpIn1. N2:17.6±0.5 (125), daf-2(e1370): 44.0±0.7 (46), daf-2(e1370); lpIn1:53.3±1.7 (39). In (A) and (B), the life span curves were plotted fromthe pooled data of the individual experiments. (C) Life span analysis ofakt-1(ok525); akt-2(ok393) and akt-1(ok525); akt-2(ok393); lpIn1.wild-type (N2), 14.9±0.4 (90); akt-1(ok525); akt-2(ok393), 34.2±0.8(100); akt-1(ok525); akt-2(ok393); lpIn1, 38.8±0.9 (90).

FIG. 17 is a graphical depiction of life span analysis showing thatoverexpression of jnk-1 shows a further life span extension onakt-1/akt-2 RNAi. Life span analysis of jnk-1 overexpression strain(lpIn1) on akt-1/akt-2 RNAi. Mean life span±standard error (total numberof animals scored): N2 on control RNAi: 16.4±0.4 days, N2 on akt-1/akt-2RNAi: 18.3±0.6 days, lpIn1 on control RNAi: 19.1±0.6 days, lpIn1 onakt-1/akt-2 RNAi: 26.5±0.7 days (P<0.0001).

FIG. 18 is an immunoblot probed with phosphor-JNK antibody (upper panel)and the same blot reprobed with JNK antibody (lower panel).

FIG. 19 is a graphical depiction of life span analysis of jkk-1;lpIn2.N2+pRF4: 15.2±0.3 (105), lpIn2: 18.8±0.5 (97), jkk-1;lpIn2: 15.0±0.3(130). The life span curves were plotted from the pooled data ofindividual experiments.

FIGS. 20A-B are immunoblots showing that JNK-1 interacts with andphosphorylates DAF-16. (A) Co-immunoprecipitation assay. Followingtransfection with either Flag-DAF-16 alone or in combination withXpress-JNK-1, cell lysates were immunoprecipitated with anti-Xpressantibody and immunoblotted with anti-Flag antibody. (B) in vitro kinaseassay. Cells were transfected with Xpress-JNK-1, activated by UV,followed by immunoprecipitation with anti-Xpress antibody and incubationwith His-DAF-16 (N-terminal fragment) in kinase buffer. Loading of thesubstrates (His-DAF-16 (N-terminal fragment) and c-Jun) is shown in theright panel stained with Coomassie Blue. In both (A) and (B), theexpression of protein was confirmed by immunoblotting with anti-Flag oranti-Xpress antibody.

FIG. 21 is an immunoblot showing that a kinase-dead JNK-1 (APF) fails tophosphorylate DAF-16. The kinase-dead JNK-1 construct was created byreplacing TPY residues with APF, and the in vitro kinase assay wasperformed using N-terminal portion of DAF-16 as a substrate. Theexpression of protein was confirmed by immunoblotting with anti-Xpressantibody.

FIGS. 22A-B present data which demonstrates that JNK-1 promotes thetranslocation of DAF-16 into the nucleus in response to heat stress. (A)Heat stress assay of jnk-1 overexpression strain (lpIn1 and lpIn2). Meansurvival time±standard error (total number of animals scored): N2+pRF4,10.8±0.2 (138); lpIn1, 15.3±0.3 (147); lpIn2, 14.4±0.2 (249). (B) DAF-16translocation assay. Following heat shock at 35° C. for 30 min, thenuclear translocation of DAF-16 was measured and the number of worms ineach category was counted (28).

FIG. 23 is a bar graph showing that jnk-1 overexpression strains exhibitincreased resistance to oxidative stress. 30˜40 young adults weretransferred to 96-well plates (5˜6 worms/well) containing 40 μl of 150mM paraquat at room temperature. Animals were tapped every 30 minutesand scored as dead when they did not respond to the platinum wire pick.Mean survival time±standard error (total number of animals scored):control (N2+pRF4), 1.7±0.1 (88); lpIn1, 3.1±0.1 (68); lpIn2, 2.7±0.1(118). This stress assay was repeated at least three times.

FIG. 24 is a bar graph showing that heat stress resistance by jnk-1overexpression is dependent on daf-16. 50 young adult worms were grownand picked onto either control RNAi plates or daf-16 RNAi plates andkept at 35° C. Animals were tapped every hour and scored as dead whenthey did not respond to the platinum wire pick. Mean survivaltime±standard error (total number of animals scored): N2+pRF4 on controlRNAi, 9.9±0.2 (60); N2+pRF4 on daf-16 RNAi, 9.3±0.1 (84); lpIn1 oncontrol RNAi, 12.0±0.2 (98); lpIn1 on daf-16 RNAi, 9.4±0.1 (100).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery of acentral role for the JNK signaling pathway in controlling longevity. Inparticular, the invention is based on the discovery that members of theJNK signaling cascade are involved in either increasing or decreasinglife span extension associated with reduction of function mutations inthe insulin-like signaling pathway genes daf-2 and age-1 in C. elegans,and that overexpression of members of the JNK signaling cascadeincreases lifespan. The invention is further based on the discovery thatJNK-1 directly interacts with and phosphorylates DAF-16 and therebymodulates DAF-16 nuclear localization.

Accordingly, the invention features whole organism-based assays for theidentification of an agent capable of enhancing longevity. In oneaspect, the invention features a method for identifying an agent capableof enhancing longevity, involving: (a) contacting an organism having aderegulated c-jun N-terminal kinse (JNK) signaling pathway with a testagent, wherein a detectable phenotype is associated with the deregulatedJNK signaling pathway; and (b) assaying for the ability of the testagent to effect the phenotype, wherein the agent is identified based onits ability to alter the phenotype as compared to a suitable control. Avariation on this method involves (a) contacting an organism furtherhaving a deregulated insulin signaling pathway, wherein said detectablephenotype is associated with said deregulated JNK signaling pathway andsaid deregulated insulin signaling pathway, and (b) assaying for theability of the test agent to effect the phenotype, wherein the agent isidentified based on its ability to alter the phenotype as compared to asuitable control.

In preferred embodiments, the deregulated JNK signaling pathway moleculeis selected from the group consisting of: UNC-16, MEK-1, JKK-1 andJNK-1, or a mammalian orthologue thereof. In further preferredembodiments, the deregulated insulin signaling pathway molecule isselected from the group consisting of DAF-2, AAP-1, IRS-1, AGE-1, PDK-1,AKT-1, AKT-1 and DAF-2, or a mammalian orthologue thereof.

In preferred embodiments the phenotypes include, but are not limited to,one or more of the following: increased or decreased life span,constitutive or defective dauer formation, increased or decreased bodysize, and increased or decreased stress resistance, e.g. oxidativestress, UV stress, hypoxic stress, heavy metal stress and heat stress.

In another aspect, the invention provides a method for identifying anagent capable of enhancing longevity, involving: (a) contacting anorganism having a c-jun N-terminal kinase (JNK) signaling pathway with atest agent, and (b) assaying for the ability of the test agent to effecta downstream indicator of the c-jun N-terminal kinase (JNK) signalingpathway, wherein the agent is identified based on its ability to alterthe indicator as compared to a suitable control. In one embodiment, theJNK signaling pathway is deregulated.

In preferred embodiments, the agent alters activity of the indicator,cellular localization, e.g. cytoplasmic to nuclear, of the indicator,and nucleic acid or polypeptide expression, e.g. rate of expression orsteady state expression. In further preferred embodiments, the agentalters the post-translational modification state of the indicator, e.g,.phosphorylation state. In still further preferred embodiments, theindicator is DAF-16 or superoxide dismutase, or a glucose transporter,e.g., glucose transporter 1 or glucose transporter 4.

In preferred embodiments, the organism is a nematode, e.g., C. elegansor a parasitic nematode.

The invention further features cell-based assays for the identificationof an agent capable of enhancing longevity. In one aspect, the inventionprovides methods for identifying an agent that enhances longevity,involving (a) contacting a cell with a test agent, said cell having aJNK signaling pathway; (b) detecting an indicator of JNK signaling; and(c) identifying the agent based on its ability to modulate JNK signalingin said cell. In another aspect, the invention provides a method foridentifying an agent that enhances longevity, comprising (a) contactinga cell with a test agent, said cell having a JNK signaling pathway andan insulin signaling pathway; (b) detecting an indicator of JNKsignaling and insulin signaling; and (c) identifying the agent based onits ability to modulate JNK signaling and insulin signaling in saidcell.

In one embodiment, the agent inhibits the signaling pathway(s), e.g.,inhibits the insulin signaling pathway and the JNK signaling pathway. Inanother embodiment, the agent enhances the JNK signaling pathway and,optionally, inhibits the insulin signaling pathway.

In preferred embodiments, the cell is a mammalian cell, e.g. a humancell, a bacterial cell, a yeast cell, or is derived from a nematode.

In preferred embodiments, the indicator is selected from, but notlimited to, one or more of the following: conversion of substrate tocorresponding product catalyzed by a downstream enzyme in the pathway,activation or inhibition of a downstream enzyme in the pathway, atranscriptional event in the pathway, and activation or inhibition of atranscription factor regulated by the pathway.

In preferred embodiments, the indication involves an endogenous gene orprotein, or a reporter gene or protein.

The invention further features an in vitro method of identifying anagent capable of enhancing longevity, involving: (a) contacting a firstassay composition with a test compound, wherein the assay compositioncomprises a JNK signaling pathway molecule; (b) detecting activity orexpression of the JNK signaling pathway molecule; (c) contacting asecond assay composition with the test compound, wherein the assaycomposition comprises an insulin signaling pathway molecule; and (d)detecting activity or expression of the insulin signaling pathwaymolecule, wherein the agent is identified based on its ability tomodulate activity or expression of the JNK signaling pathway moleculeand insulin signaling pathway molecule.

In preferred embodiments, the deregulated JNK signaling pathway moleculeis selected from the group consisting of: UNC-16, MEK-1, JKK-1 andJNK-1, or a mammalian orthologue thereof. In further preferredembodiments, the deregulated insulin signaling pathway molecule isselected from the group consisting of DAF-2, AAP-1, IRS, AGE-1, PDK-1,AKT-1, AKT-1 and DAF-2, or a mammalian orthologue thereof.

In further preferred embodiments, the assay composition is a cell-freeextract, or purified proteins.

The invention further features a method for enhancing longevity in asubject, involving: (a) selecting a subject in need of enhancedlongevity; and (b) administering to the subject a pharmacologicallyeffective dose of an agent that modulates a JNK signaling pathwaymolecule, wherein the modulation of the JNK signaling pathway moleculein the subject enhances longevity.

In preferred embodiments, the agent modulates a JNK signaling pathwaymolecule selected from, but not limited to, the group consisting of:MAPKKKs, JNK, MKK4, MKK7 and JIF scaffold protein. In one embodiment,said agent increases the activity or expression of JNK. In anotherembodiment, said agent inhibits JNK. In further preferred embodiments,the agent inhibits an insulin signaling pathway molecule selected from,but not limited to, the group consisting of IR, IGF, IRS, PI3-K, PTENphosphatase, PDK1, PKB and forkhead transcription factors, e.g. FKHR.

In further preferred embodiments, the subject is an aging or agedsubject, a subject exhibiting at least one symptom of premature aging,or has an aging-associated disorder.

So that the invention may be more readily understood, certain terms arefirst defined.

“Longevity” and “life-extension”, used interchangeably herein, alsoinclude delay and/or stabilizing the aging process. Preferably, thelongevity is due to an extension of the mature life phase, as opposed toan extension of the immature life phase (i.e., delay in maturity).

A “function” of a polynucleotide can be on any level, including DNAbinding, transcription, translation, processing and/or secretion ofexpression product, interaction (such as binding) of expression productwith another moiety, and regulation (whether repression orde-repression) of other genes. It is understood that a life-extensionpolynucleotide or polypeptide includes fragments, or regions, of apolynucleotide or polypeptide, as long as the requisite life-extensionphenotype is observed.

The term “in vitro” has its art recognized meaning, e.g., involvingpurified reagents or extracts, e.g., cell extracts. The term “in vivo”also has its art recognized meaning, e.g., involving living cells, e.g.,immortalized cells, primary cells, cell lines, and/or cells in anorganism.

A “gerontogene” is a gene, the alteration of which slow aging, extendslifespan and/or enhances late-life health. See e.g., Rattan (1985)Bioessays 2:226-228. Such genes can also been termed “longevityassurance genes” or “longevity associated genes” (both abbreviated“LAGs”). See e.g., D'Mello et al., (1994) J. Biol. Chem.269:15451-15459.

The term “JNK signaling pathway” refers to the signaling pathwayinvolving proteins (e.g., enzymes) and other non-protein molecules(e.g., precursors, substrates, intermediates or products), utilized intransmission of an intracellular signal from a cell membrane (e.g., froma cell surface receptor) to the nucleus, wherein such signaltransmission involves at least c-jun N-terminal kinase (JNK). FIG. 1includes a schematic representation of the JNK signaling pathway.Additional signaling molecules in the JNK signaling pathway in mammals,for example, include the MAP Kinase Kinase Kinases (MAPKKKs), JNK, MKK4,MKK7, and JIF scaffold protein. Such signaling molecules in C. elegans,for example, include, UNC-16, MEK-1, JKK-1, and c-jun N-terminalkinase-1 (JNK-1), (and the corresponding genes encoding these molecules,i.e., unc-16, mek-1, jkk-1 and jnk-1, respectively.

The term “insulin signaling pathway” (or “insulin-like signalingpathway”) refers to the signaling pathway involving proteins (e.g.,enzymes) and other non-protein molecules (e.g., precursors, substrates,intermediates or products), utilized in transmission of an intracellularsignal from a cell membrane to the nucleus, in particular, from aninsulin receptor (IR) or insulin-like growth factor (IGF) receptor atthe cell surface to the nucleus. Additional signaling molecules in theinsulin signaling pathway in mammals, for example, include insulinreceptor substrate (IRS), phosphatidylinositol 3-kinase (PI3-K), PTENphosphatase, phosphoinositide kinase 1 (PDK1), protein kinase B (PKB)and forkhead transcription factors (FKHR). Such signaling molecules inC. elegans, for example, include IST-1, DAF-2, AAP-1, AGE-1, PDK-1,AKT-1, DAF-18 and DAF-16 (and the corresponding genes encoding thesemolecules, i.e., ist-1, daf-2, aap-1, age-1, pdk-1, akt-1, akt-2,daf-18, and daf-16, respectively. FIG. 2 includes a schematicrepresentation of the insulin signaling pathway.

The term “deregulated” or “deregulation” includes the alteration ormodification of at least one molecule in a signaling pathway, such thatsignal transmission by the pathway is altered or modified. Preferably,the activity or expression of at least one enzyme in the pathway isaltered or modified such that signal transmission by the pathway isaltered or modified.

The term “upmodulated” refers to an increase or enhancement of theactivity or expression of a signaling pathway molecule. The term“downmodulated” refers to a decrease or inhibition of the activity orexpression of a signaling pathway molecule.

“Impaired JNK signaling” refers to genetic or other alterations thatlead to reduced activity in the JNK signaling pathway in mammals,organisms, cells, etc. These alterations include, but are not limitedto, inhibition of expression or activity of signaling molecules involvedin insulin signaling in mammals, organisms, cells, etc.

“Impaired insulin signaling” refers to genetic or other alterations thatlead to reduced activity in the insulin or insulin-like signalingpathway in mammals, organisms, cells, etc. These alterations include,but are not limited to, inhibition of expression or activity ofsignaling molecules involved in insulin signaling in mammals, organisms,cells, etc.

“Increased activity” or “enhanced activity” of a signaling molecule, forexample, DAF-16 or a DAF-16 orthologue, refers to increased daf-16 ordaf-16 orthologue transcription or translation, increased DAF-16 orDAF-16 orthologue activation and/or increased target protein activation.

A “target protein” of DAF-16 or a DAF-16 orthologue refers to anyprotein that DAF-16 or a DAF-16 orthologue either binds to directly inorder to modulate, or whose transcription or translation is modulated bybinding of DAF-16 or a DAF-16 homolog to the regulatory region of thegene or the mRNA encoding the protein. Target proteins can include, butare not limited to, HSP70, HSP90, catalase, ubiquitin and/or superoxidedismutase.

“Candidate agents” or “candidate molecules” means agents or moleculesthat can be tested in screening assays for suitability as agents toextend life span. Typically, candidate agents are small molecules,peptides, oligonucleotides and/or derivatives thereof, or othercompounds known to be useful as screening candidates in the drugdiscovery field.

As used herein, the term “agent” means a biological or chemical compoundsuch as a simple or complex organic or inorganic molecule, a peptide,protein, oligonucleotide, polynucleotide, carbohydrate, or lipoprotein.A vast array of compounds can be synthesized, for example oligomers,such as oligopeptides and oligonucleotides, and synthetic organiccompounds based on various core structures, and these are also includedin the term “agent”. In addition, various natural sources can providecompounds for screening, such as plant or animal extracts, and the like.Compounds can be tested singly or in combination with one another.

As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and translated into peptides,polypeptides, or proteins.

An agent that “modulates” life-extension is an agent that affectslife-extension, or lifespan, whether directly or indirectly, whethernegatively or positively.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Signaling Pathways

In mammalian insulin signaling, many signaling pathways operatedownstream of the insulin receptor. One such pathway, themitogen-activated protein kinase (MAPK) signaling pathway, is involvedin regulating normal development, mitogenesis, and various stressresponses (Kido Y. et al. (2001) J of Clin Endocrin and Met 86:972-979).MAPKs can be divided into three groups: the c-Jun N-terminal (JNK orstress-activated protein kinase); the extracellular signal-regulatedkinase (ERK); and p38 kinases. MAPKs are activated via dualphosphorylation of threonine and tyrosine residues in their activationloops. The specific MAPK kinases (MAPKKs or MEKs) that carry out thisreaction are themselves phosphorylated and activated by specific MAPKKkinases (MAPKKKs).

In the JNK signaling cascade (reviewed in Ip Y. T. & Davis R. J. (1998)Curr Opin Cell Biol 10:205-219; Weston C. R. & Davis R. J. (2002)12:14-21), JNK activity is strongly stimulated in vertebrate cellculture by inhibitors of protein biosynthesis, such as cycloheximide andanisomycin, by inflammatory cytokines, such as tumor necrosisfactor-alpha (TNF-alpha) and interleukin-1B (IL-1B), and by heat,osmotic shock, UV light or other DNA-damaging agents. When JNK isactivated, it migrates from the cytoplasmic compartment into the nucleusand stimulates the activity of several transcription factors includingc-Jun, ATF-2, ELK-1 and p53. In mammalian cells, the JNK signal cascadebegins with the stress-induced activation of MEKK4. MEKK4 in turnactivates the two MAPKKs, MKK4 and MKK7, both of which are believed toactivate JNK. While MKK4 also functions as an activator of p38, MKK7probably functions as a specific activator of JNK (Cavigelli et al.(1995) EMBO J. 14:5957-5964; Derijard et al. (1995) Science 267:682-685;Tournier et al. (1997) PNAS USA 94:7337-7342). Finally, JNK in turnactivates c-Jun.

In C. elegans, jnk-1 is the jnk homolog, mek-1 is highly homologous tomammalian mkk7, and jkk-1 has 41.6% identity with MKK7 in the kinasedomain (Kawasaki et al. (1999) EMBO J. 18:3604-3615; Koga et al. (2000)EMBO J. 19:5148-5156). Both jnk-1 and jkk-1 are co-expressed in the cellbodies and the axons of most neurons (Kawasaki et al. (1999) EMBO J.18:3604-3615), while mek-1 is expressed in pharyngeal muscles, uterus, aportion of intestine and neurons in the ring, ventral and anal ganglia(Koga et al. (2000) EMBO J. 19:5148-5156). Although individual jnk-1,jkk-1 or mek-1 mutants do not show developmental defects, disruption ofjkk-1 or jnk-1 results in defective body movement coordination viatype-D GABAergic motor neurons. In addition, disruption of jnk-1 ormek-1 results in hypersensitivity to heavy metals, such as copper andcadmium ions (Koga et al. (2000) EMBO J. 19:5148-5156; Kawasaki et al.(1999) EMBO J. 18:3604-3615; Villanueva, A. et al. (2001) EMBO J.20:5114-5128).

The C. elegans genome also contains mkk-4, which is highly homologous tomammalian mkk4. Inactivation of mkk-4 only resulted in an egg-layingdefect in adult hermaphrodites, and thus this mutant did not display asimilar phenotype to that of the other JNK pathway members (VillanuevaA. et al. (2001) EMBO J. 20:5114-5128). Finally, the most recent memberof the JNK signaling pathway to be identified in C. elegans is the geneunc-16. The gene unc-16 encodes a JIF scaffold protein homolog (Byrd D.T. et al. (2001) Neuron 32:787-800). The function of JIF proteins is toact to gather several of the components of the JNK pathway, includingMKK7 and JNK, and in this way eases signal transmission by the signalingpathway (Yasuda J. et al. (1999) Mol and Cell Biol 19:7245-7254).

Prior to the instant invention, there was no knowledge of a role for theJNK signaling pathway in longevity. The instant inventors firstperformed life span analysis in various C. elegans strains havingmutations in various JNK signaling pathway molecules. The JNK family waschosen as a potential pathway that could act downstream of daf-2 ininsulin-like signaling (see background) for the following reasons:

-   -   (1) The JNK pathway is activated by the stress response, and        daf-2 and age-1 mutants are known to be resistant to stresses,        such as heat, oxidative damage and heavy metals (Lithgow G. J.        et al. (1994) J. Gerontol. 49:B270-276; Lithgow, G. J., et        al. (1995) PNAS USA 92:7540-4; Murakami S. &        Johnson T. E. (1996) Genetics 143:1207-1218; Honda Y. &        Honda S. (1999) FASED J 13:1385-1393; Baryste D. et al. (2001)        FasEB J 15:627-634; Friedman D. B. & Johnson T. E. (1988)        Genetics 118:75-86; Gems D. et al. (1998) Genetics 150:129-155).    -   (2) Several genes in the JNK signaling pathway, including jnk-1        and the two MKK4 homologs, jkk-1 and mek-1, have already been        isolated in C. elegans. These genes have been shown to affect        resistance to stress; reducing the activity of any of these        three genes yielded worms that displayed defective body movement        coordination and/or hypersensitivity to heavy metal stresses        (Villanueva A. et al. (2001) EMBO J 20:5114-5128; Koga M. et        al. (2000) EMBO J 19:5148-5156; Kawasaki M. et al. (1999) EMBO J        18:3604-3615). This is relevant because both daf-2 and age-1        mutants are resistant to heavy metals and many other types of        stress (reviewed in Kimura K. et al. (1997) Science        277:942-946). A strong correlation has been made between life        span extension and stress resistance (Lithgow G. J. and        Walker G. A. (2002) Mech of Aging and Dev. 123:765-771). A very        recent publication has shown that heterozygous knockout mice for        IGF-R have extended life span and increased resistance to        oxidative stress (Holzenberger M. et al. (2002) Nature, in        press.), indicating that IGF-1R may be a central regulator of        lifespan in mammals. While only one insulin-like receptor        homolog, DAF-2, has been clearly identified in C. elegans, a        very recent publication has reported a new family of putative        insulin receptor-like proteins (Dlakic M., (2002) Curr. Biol        12(5) R155-R157). The correlation between life span extension        and stress resistance suggests that the JNK signaling genes        could play a role in life span regulation.    -   (3) In mammals, JNK interacts with the insulin receptor        substrate (IRS) molecule and phosphorylates IRS at Ser307 in        vitro and in vivo in mice (Aguirre V. et al. (2000) J Biol Chem        275: 9047-9054; J. Hirosumi et al. (2002) Nature 420:333-336).        IRS is an adaptor molecule through which insulin receptor        signals, both in mammalian systems and Drosophila. In obese        mammals, TNF-alpha or FFA produced in adipose tissue activates        JNK. Activated JNK in turn phosphorylates IRS at Ser307, causing        an inhibition of IRS activity and thereby inhibiting insulin        signaling. In this way, JNK promotes the development of insulin        resistance that is associated with obesity and type 2 diabetes        (J. Hirosumi et al. (2002) Nature 420:333-336). Very recent        publications have identified the C. elegans homolog of IRS,        named IST-1, along with AAP-1, the C. elegans homolog of the        p50/p55 subunits of PI3-K (Wokow C. A. et. al. (2002) J. Biol.        Chem. 277:49591-49597).

Based on a detailed life span analysis in the various C. elegansmutants, it was discovered that reduction of function mutations in theJNK pathway cause either a reduction of life span or no change in lifespan in wild type background. The mutations were then placed incombination with a reduction of function mutation in the insulin-likereceptor gene daf-2 or the downstream PI-3-kinase age-1. The resultingdouble mutant causes either a synergy in the life span (greater thaneither mutant alone) or a reduction of life span (intermediate: lowerthan the daf-2 or age-1 mutation alone but not quite down to the levelof the JNK mutation on its own). Remarkably, even mutations that did noteffect life span on their own, show an effect when placed in combinationwith another mutation that causes a change in levels of the insulinpathway (and therefore daf-16). In further experiments, the creation ofjnk-1 overexpression mutants and life span analysis revealed that anincrease in jnk-1 expression significantly extends life span.Biochemical and genetic data showed that JNK-1 directly interacts withand phosphorylates DAF-16, and that JNK-1 thereby promotes thetranslocation of DAF-16 to the nucleus in response to heat stress.

Based on these findings, novel screening assays have been developed forthe identification of longevity-promoting agents (e.g., anti-agingagents), as well as therapeutic methods for increasing longevity and/orquality of life in aging individuals which feature targeting the JNKsignaling pathway in such individuals. These features of the instantinvention are described in detail in the following subsections.

II. Screening Assays

The methods of the invention are suitable for use in methods to identifyand/or characterize potential pharmacological agents, e.g. identifyingnew pharmacological agents from a collection of test substances, inparticular, pharmacological agents for use in increasing life spanand/or enhancing quality of life in aged individuals. Pharmacologicalagents identified according to the methodologies of the invention arealso useful, for example, in enhancing stress resistance in individuals,and increasing the cytoprotective abilities of cells.

Pharmacological agents identified according to the methodologies of theinvention are additionally useful in preventing or reducing obesity inindividuals who suffer from obesity or in individuals who arepredisposed to developing obesity, e.g., by reducing the adiposity ofcells. Pharmacological agents idenetified according to the methodologiesof the invention are also useful in treating individuals suffering fromtype II diabetes and preventing the onset of type II diabetes inindividuals at risk of developing type II diabetes. Pharmacologicalagents are also useful in promoting apoptosis in cells, e.g., promotingapoptosis in tumor cells in individuals undergoing cancer therapies,e.g., cancer chemotherapy or radiation therapy. Pharmacological agentsare additionally useful in inhibiting or downmodulating apoptosis incells, e.g., in cells during development.

The methods described herein are in vitro and in vivo cell- and animal(e.g., nematode)-based screening assays.

A. Screening in Whole Organisms

The invention provides screening assays in whole organisms. In the wholeorganism-based embodiments, whole organisms comprising the organismhaving a deregulated JNK signaling pathway and/or insulin signalingpathway are used for testing agents. In a particular embodiment, thederegulated JNK signaling pathway is downmodulated. In anotherembodiment, the deregulated JNK signaling pathway is upmodulated.

The invention provides a method for identifying an agent capable ofenhancing longevity, comprising: (a) contacting an organism having aderegulated c-jun N-terminal kinse (JNK) signaling pathway with a testagent, wherein a detectable phenotype is associated with the deregulatedJNK signaling pathway; and (b) assaying for the ability of the testagent to effect said phenotype, wherein the agent is identified based onits ability to alter said phenotype as compared to a suitable control. Avariation on this method comprises (a) contacting an organism furtherhaving a deregulated insulin signaling pathway, wherein said detectablephenotype is associated with said deregulated JNK signaling pathway andsaid deregulated insulin signaling pathway, and (b) assaying for theability of the test agent to effect said phenotype, wherein the agent isidentified based on its ability to alter said phenotype as compared to asuitable control.

In one embodiment, the deregulated insulin signaling pathway molecule isselected from the group consisting of DAF-2, IST-1, AAP-1, AGE-1, PDK-1,AKT-1, AKT-2 and DAF-18, or a mammalian orthologue thereof. In anotherembodiment, the deregulated JNK signaling pathway molecule is selectedfrom the group consisting of (UNC-16), (MEK-1), (JKK-1), and c-junN-terminal kinse-1 (JNK-1), or a mammalian orthologue thereof.

In a preferred embodiment of the invention, the roundworm Caenorhabditiselegans is employed. C. elegans is a simple soil nematode species thathas been extensively described at the cellular and molecular level, andis a model organism for biological studies. C. elegans can developthrough a normal life cycle that involves four larval stages and a finalmolt into an adult hermaphrodite. The dauer pathway is an alternativelife cycle stage common to many nematode species which is normallytriggered by environmental stresses such as starvation, temperatureextremes, or overcrowding. Genetically, the dauer pathway has been mostintensively studied in C. elegans. The response to overcrowding in C.elegans is mediated by a substance known as dauer pheromone, which issecreted by the animals. When dauer pheromone becomes sufficientlyconcentrated, it triggers commitment to the dauer alternative life cyclestage. A strong correlation exists between a constitutive dauer and thelong-lived phenotype.

In preferred embodiments of the invention, the detectable phenotype isincreased or decreased life span. In another embodiment, the detectablephenotype is constitutive dauer formation or defective dauer formation.In other embodiments, the phenotype is increased or decreased body size,or increased or decreased stress resistance, wherein stress resistanceis selected from, but not limited to, the group consisting of oxidativestress, ultraviolet (UV) stress, hypoxic stress, heavy metal stress andheat stress.

When screening for an effect of dauer formation, the assay population ofC. elegans is preferably exposed to test agent during the portion of thelife cycle at which commitment to the dauer pathway is made. Measurementof dauer formation has been previously described. See e.g., Riddle etal., Genetic and Environmental Regulation of Dauer Larva Development, InRiddle, Blumenthal, Meyer, and Priess (eds), C. ELEGANS II., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1997). In mutantstrains containing deregulated JNK and/or insulin signaling andexhibiting a constitutive dauer phenotype, an agent is identified basedon its ability to reverse that phenotype.

Life span assays have also been well described (Apfeld J. & Kenyon C.(1998) Cell 95: 199-210). In strains that exhibit an extended life spanphenotype, an agent is identified based on its ability to either furtherextend or shorten the lifespan. Resistance to ultraviolet (UV) stress isdetermined by exposing the organism to UV light and measuring life spanfrom the day of UV treatment. Oxidative stress resistance is determinedby exposing the animals to paraquat, which produces superoxide whentaken up by cells, and determining survival from the day of treatment(Feng et al. (2001) Dev. Cell 1:1-20.). Heat tolerance is measured byexposing adult animals to a 35° C. heat shock for 24 hours, and thenscoring the animals for viability.

In assay formats featuring indicator phenotypes, the phenotype of theanimals may be detected by direct observation. An alternative to directobservation is mechanical detection of the animals. For instance, suchdetection could involve the determination of optical density across thetest surface by a machine. The animals would be detected by changes indensity at the location where an animal was located. Alternatively, ifthe animals are expressing a reporter gene that can be detected inliving animals (e.g., GFP), a machine could monitor the animals using asuitable reporter gene detection protocol.

If desired, additional tests may be conducted using the agent identifiedto further characterize the nature of the agent's function with respectto longevity. For example, egg laying may also be measured to determinewhether the longevity occurs by delaying maturity. As another example,other phenotypes associated with other gerontogenes could be tested todetermine whether the identified agent affects functional pathwaysassociated with these other genes.

Another embodiment of the invention provides a method for identifying anagent capable of enhancing longevity, comprising: (a) contacting anorganism having a c-jun N-terminal kinse (JNK) signaling pathway with atest agent; (b) assaying for the ability of the test agent to effect adownstream indicator of said c-jun N-terminal kinse (JNK) signalingpathway, wherein the agent is identified based on its ability to altersaid indicator as compared to a suitable control. In one embodiment, theJNK signaling pathway is deregulated.

In such assays, the organism is a nematode. In a preferred embodiment,the nematode is C. elegans. In a further embodiment, the organism is aparasitic nematode. In one embodiment, the organism is not an insect. Inyet another embodiment, the deregulated signaling molecule in said assayis selected from the group consisting of (UNC-16), (MEK-1), (JKK-1), andc-jun N-terminal kinse-1 (JNK-1).

In another embodiment, the indicator is selected from, but not limitedto, the group consisting of DAF-16, superoxide dismutase (SOD), glucosetransporter 4 (GLUT4) and glucose transporter 1 (GLUT1). In a preferredembodiment, the indicator is DAF-16 or a mammalian orthologue thereof.Recent publications indicate that two other members of the insulin-likesignaling pathway in C. elegans, DAF-9 and DAF-12, function downstreamof DAF-16 (Gerisch B. et al. (2001) Dev. Cell, 1(6):841-51; Jia K. etal. (2002) Development 129:221-231). In C. elegans, daf-9 encodes acytochrome P450 related to vertebrate steroidogenic hydroxylases,suggesting it could metabolize a DAF-12 ligand. In another embodiment,therefore, the indicator may be either DAF-9 or DAF-12.

In such an assay, the agent may be identified based on its ability toincrease or decrease the indicator. The agent may alter expression ofthe indicator, wherein the expression is nucleic acid expression orpolypeptide expression. The alteration of expression may be a change inthe rate of expression or steady state expression.

In one embodiment, the agent alters the activity of the indicator. In apreferred embodiment, the agent may alter the post-translationalmodification state of the indicator, e.g. the phosphorylation state ofthe indicator, e.g., the phosphorylation state of DAF-16 or a mammalianorthologue thereof. In a particularly preferred embodiment, theindicator is the phosphorylation state of DAF-16 at specificphosphorylation sites in DAF-16, or a mammalian orthologue thereof, thatare phosphorylated by the kinase activity of JNK-1 or a mammalianorthologue thereof. In one embodiment, DAF-16 phosphorylation by JNK-1is upmodulated. In one embodiment, DAF-16 phosphorylation by JNK-1 isdownmodulated. The specific phosphorylation sites in DAF-16 or itsmammalian orthologues, e.g., FOXO, can be determined using methodologieswell known to one of ordinary skill in the art and, for example, asdescribed in the Examples herein. Techniques are well known in the artfor analyzing phosphorylation and other post-translational modificationstates. For example, phosphorylation may be determined by the use ofantibodies to phospho-epitopes to detect a phosphorylated polypeptide byWestern analysis.

In another embodiment, the agent may alter the cellular localization ofthe indicator, such as from cytoplasmic to nuclear. In a preferredembodiment, the agent alters the nuclear translocation of DAF-16 or amammalian orthologue thereof, e.g., the nuclear translocation of DAF-16regulated by jnk-1, e.g, in response to stress. In one embodiment,nuclear translocation of DAF-16 is upmodulated. In one embodiment,nuclear translocation of DAF-16 is downmodulated. Changes in cellularlocalization can be determined by introducing a chimeric form of theindicator containing a reporter gene. Plasmid constructs can beintroduced into C. elegans using described transformation methods. Seee.g., Mello et al., (1991) EMBO J. 10:3959-3970. Preferably, the plasmidconstructs are linear constructs. An important aspect of transformationin C. elegans is that plasmid constructs can be easily cotransformed,thus allowing for assay formats in which C. elegans are engineered toexpress, for example, non-C. elegans signaling pathway molecules andreporter genes. Preferably, a reporter gene is used that can be scoredin a living animal, but does not affect the indicator phenotype of theanimal. For example, green fluorescent protein (herein referred to as“GFP”) is a widely used reporter molecule in living systems. Ellenberg(1999) Trends Cell Biol. 9:52-56; Chalfie et al., (1994) Science263:802-805.

B. Cell-Based Screening Assays

The invention further features cell-based assays for the identificationof an agent capable of enhancing longevity. In one embodiment, theinvention provides methods for identifying an agent that enhanceslongevity, comprising (a) contacting a cell with a test agent, said cellhaving a JNK signaling pathway; (b) detecting an indicator of JNKsignaling; and (c) identifying the agent based on its ability tomodulate JNK signaling in said cell. The invention further provides amethod for identifying an agent that enhances longevity, comprising (a)contacting a cell with a test agent, said cell having a JNK signalingpathway and an insulin signaling pathway; (b) detecting an indicator ofJNK signaling and insulin signaling; and (c) identifying the agent basedon its ability to modulate JNK signaling and insulin signaling in saidcell.

The cell-based screening assays described herein have several advantagesover conventional drug screening assays: 1) if an agent must enter acell to achieve a desired therapeutic effect, a cell-based assay cangive an indication as to whether the agent can enter a cell; 2) acell-based screening assay can identify agents that, in the state inwhich they are added to the assay system are ineffective to modulate theJNK and/or insulin signaling polynucleotide and/or polypeptide function,but that are modified by cellular components once inside a cell in sucha way that they become effective agents; 3) most importantly, acell-based assay system allows identification of agents affecting anycomponent of a pathway that ultimately results in characteristics thatare associated with JNK and/or insulin signaling polynucleotide and/orpolypeptide function.

In one embodiment, the agent is identified based on the ability toinhibit JNK signaling. In a preferred embodiment, the agent isidentified based on the ability to inhibit JNK signaling andadditionally to inhibit insulin signaling. In another prefferedembodiment, the agent is identified based on the ability to enhance JNKsignaling. In another embodiment, the agent is identified based on theability to enhance JNK signaling and inhibit insuling signaling. Inpreferred embodiments, the indicator is altered cellular localization ofDAF-16 or a mammalian orthologue thereof, e.g., FOXO, e.g., nuclearlocalization of DAF-16 or a mammalian orthologue thereof, e.g., FOXO. Inother preferred embodiments, the indicator is altered phosphorylationstate of DAF-16 or a mammalian orthologue thereof, e.g., phosphorylationof DAF-16 by JNK-1.

In one embodiment, suitable host cells include, but are not limited to,fungi (including yeast), bacterial, insect and mammalian. In a preferredembodiments, the host cell is a human cell or is derived from anematode. In one embodiment, the cell is not an insect cell.

An indicator of the JNK signaling and/or insulin signaling may include aJNK signaling and/or insulin signaling polynucleotide and/orpolypeptide. Characteristics associated with said JNK signaling and/orinsulin signaling polynucleotide and/or polypeptide depend upon thepolynucleotide or polypeptide. Functional characteristics include, butare not limited to, transcription, translation (including levels ofprecursor and/or processed polypeptide), location of protein product(such as nuclear or membrane localization), post-translationalmodification of protein product (such as phosphorylation oracetylation), any enzymatic activities, such as kinase activity,structural and/or functional phenotypes (such as stress resistance orlife cycle), and expression (including repression or de-repression) ofany other genes known to be controlled (modulated) by thepolynucleotide. Any measurable change in any of these and otherparameters indicate that the agent may be useful. In a preferredembodiment, given that JNK and/or insulin signaling pathway moleculesthat regulate longevity have been identified by their ability to conferlife extension when their function is reduced, useful agents willpreferably be agents that confer decreased functionality. In anotherpreferred embodiment, given that overexpression of JNK signaling pathwaymolecules, e.g., JNK, have been identified to confer life extension,useful agents will additionally preferably be agents that conferincreased activity or expression, e.g., overexpression, of JNK signalingpathway molecules. In particularly preferred embodiments, given that JNKhas been identified to confer life extension by phosphorylating DAF-16and thereby modulating DAF-16 translocation to the nucleus, usefulagents will preferably be agents that increase kinase activity of JNK-1or a mammalian orthologue thereof for DAF-16 or a mammalian orthologuethereof. In other preferred embodiments, given that JNK has beenidentified to phosphorylate DAF-16 and thereby modulate DAF-16translocation to the nucleus, and given that DAF-16/FOXO promotesapoptosis, useful agents will preferably be agents that increase kinaseactivity of JNK for DAF-16/FOXO to thereby promote apoptosis in cells.In other embodiments, useful agents will preferably be agents thatdecrease kinase activity of JNK for DAF-16/FOXO to thereby downmodulateapoptosis in cells.

Modulation of function of a JNK signaling pathway molecule,polynucleotide and/or polypeptide, may occur at any level. An agent maymodulate function by reducing or preventing transcription of a JNKsignaling pathway polynucleotide. An example of such an agent is onethat binds to the upstream controlling region, including apolynucleotide sequence or polypeptide. An agent may modulatetranslation of mRNA. An example of such an agent is one that binds tothe mRNA, such as an anti-sense polynucleotide, or an agent whichselectively degrades or stabilizes the mRNA. An agent may modulatefunction by binding to the JNK signaling pathway polypeptide. An exampleof such an agent is a polypeptide or a chelator.

In preferred embodiments, to identify agents that inhibit JNK and/orinsulin signaling, the skilled artisan could look for conversion of asubstrate to the corresponding product catalyzed by a downstream enzymein the signaling pathway. The artisan could look for activation orinhibition of a downstream enzyme in the pathway, for example theactivation of downstream kinase in the JNK signaling pathway. Theartisan could further look for an alteration of a transcriptional eventregulated by the pathway, such as the expression of a nuclear factorregulated by the pathway. Another indicator may be the activation orinhibition of a transcription factor regulated by the pathway. In eachof these instances, the indication may involve an endogenous gene orprotein. Alternatively, the indication could involve a reporter gene orprotein.

Measuring all of these parameters (such as those using reporter genes)involve methods known in the art and need not be discussed herein. Forexample, degree of transcription can be measured using standard Northernanalysis. Amount of expression product may be measured simply by Westernanalysis (if an antibody is available) or by a functional assay thatdetects the amount of protein, such as kinase activity.

Cell-based screening assays of the present invention can be designed,e.g., by constructing cell lines or strains of animals in which theexpression of a reporter protein, i.e., an easily assayable protein,such as β-galactosidase, chloramphenicol acetyltransferase (CAT), greenfluorescent protein (GFP) or hiciferase, is dependent on JNK and/orinsulin signaling polynucleotide and/or polypeptide function. The cellis exposed to a test agent, and, after a time sufficient to effectβ-galactosidase expression

and sufficient to allow for depletion of previously expressedβ-galactosidase, the cells are assayed for the production ofβ-galactosidase under standard assaying conditions.

Reporter genes include, but are not limited to, alkaline phosphatase,chloramphenicol acetyl transferase, galactosidase, luciferase and greenfluorescent protein. Identification methods for the products of reportergenes include, but are not limited to, enzymatic assays and fluorimetricassays. Reporter genes and assays to detect their products are wellknown in the art and are described, for example in Current Protocols inMolecular Biology, eds. Ausubel et al., Greene Publishing andWiley-Interscience: New York (1987) and periodic updates. Reportergenes, reporter gene assays and reagent kits are also readily availablefrom commercial sources (Stratagene, Invitrogen and etc.).

Introduction of JNK and/or insulin signaling polynucleotides (orreporter gene polynucleotides) depend on the particular host cell usedand may be by any of the many methods known in the art, such asmicroinjection, spheroplasting, electroporation, CaCl, precipitation,lithium acetate treatment, and lipofectamine treatment.

Polynucleotides introduced into a suitable host cell(s) arepolynucleotide constructs comprising a JNK and/or insulin signalingpolynucleotide. These constructs contain elements (i.e., functionalsequences) which, upon introduction of the construct, allow expression(i.e., transcription, translation, and post-translational modifications,if any) of JNK and/or insulin signaling polypeptide amino acid sequencein the host cell. The composition of these elements will depend upon thehost cell being used. For introduction into C. elegans, polynucleotideconstructs will generally contain the JNK and/or insulin signalingpolynucleotide operatively linked to a suitable promoter and willadditionally contain a selectable marker such as rol-6 (su1006). Othersuitable host cells and/or whole animals include, for example, insect,yeast and mammalian cells. In one embodiment, the host cells and/orwhole animals are not insects, e.g., Drosophila. Suitable selectablemarkers for nematode cells are those that enable the identification ofcells that have taken up the nucleic acid, such as morphologic andbehavioral markers such as rol-6 or visual markers such as greenfluorescent protein. Screening of the transfectants identifies cells oranimals that have taken up and express the polynucleotide.

In some embodiments, a JNK and/or insulin signaling polynucleotide isoperatively linked to an inducible promoter. Use of an induciblepromoter provides a means to determine whether the agent is acting via apathway involving the JNK and/or insulin signaling polynucleotide. If anagent modulates a functional characteristic of a JNK and/or insulinsignaling polynucleotide and/or polypeptide in a cell in which theinducible promoter is activated, an observation that the agent fails toelicit the same result in a cell in which the inducible promoter is notactivated indicates that the agent is affecting at least one step oraspect of JNK and/or insulin signaling polynucleotide function.Conversely, if the functional characteristic is also observed in a cellin which the inducible promoter is not activated, then it can be assumedthat the agent is not necessarily acting solely via the JNK and/orinsulin signaling polynucleotide functional pathway.

In a particularly preferred embodiment, the invention also provides amethod for identifying an agent capable of modulating the interactionbetween JNK-1 or a mammalian ortholog thereof and DAF-16 or a mammalianortholog thereof, comprising contacting a cell with a test agent, saidcell expressing JNK-1 or a mammalian ortholog thereof and DAF-16 or amammalian ortholog thereof; detecting an indicator of the interactionbetween JNK-1 or a mammalian ortholog thereof and DAF-16 or a mammalianortholog thereof, wherein an agent is identified based on its ability tomodulate the interaction between JNK-1 or a mammalian ortholog thereofand DAF-16 or a mammalian ortholog thereof.

In a preferred embodiment the interaction is a physical interaction. Inanother preferred embodiment, said interaction is the phosphorylation byJNK-1 or a mammalian orthologue thereof of DAF-16 or a mammalianorthologue thereof.

In one embodiment, the agent inhibits said interaction. In anotherembodiment, the agent promotes said interaction. The invention alsoprovides a novel agent identified according to any of these cell-basedmethods.

C. In Vitro Screening Assays

In the in vitro embodiments, an agent is tested for its ability tomodulate activity or expression of a JNK signaling pathway and,optionally, additionally an insulin signaling pathway molecule using themethods described herein.

The invention provides an in vitro method of identifying an agentcapable of enhancing longevity, comprising (a) contacting an assaycomposition with a test compound, wherein the assay compositioncomprises a JNK signaling pathway molecule; (b) detecting activity orexpression of the JNK signaling pathway molecule, wherein the agent isidentified based on its ability to modulate activity or expression ofthe JNK signaling pathway molecule.

The invention further provides an in vitro method of identifying anagent capable of enhancing longevity, comprising (a) contacting a firstassay composition with a test compound, wherein the assay compositioncomprises a JNK signaling pathway molecule; (b) detecting activity orexpression of the JNK signaling pathway molecule; (c) contacting asecond assay composition with the test compound, wherein the assaycomposition comprises an insulin signaling pathway molecule; and (d)detecting activity or expression of the insulin signaling pathwaymolecule, wherein the agent is identified based on its ability tomodulate activity or expression of the JNK signaling pathway moleculeand insulin signaling pathway molecule.

In one embodiment, the JNK signaling pathway molecule may be selectedfrom the group consisting of UNC-16, MEK-1, JKK-1, and c-jun N-terminalkinse-1 (JNK-1), or a mammalian orthologue thereof. In anotherembodiment, the insulin signaling pathway molecule may be selected fromthe group consisting of DAF-16, DAF-2, IST-1, AAP-1, AGE-1, PDK-1,AKT-1, AKT-2 and DAF-18, or a mammalian orthologue thereof.

In such an assay, the JNK signaling molecule and insuling signalingmolecule may be polynucleotide(s) or polypeptide(s). In such an assay,the JNK signaling molecule and insuling signaling molecule may bepresent as part of a cell-free extract or a partially purified system.Alternatively, they may be purified or recombinant. The signalingpathway molecules to be used in these screening methods may be obtainedusing standard synthetic methods known in the art, including, but notlimited to, isolation from natural sources, recombinant methods,chemical synthetic methods, and enzymatic digestion followed bypurification.

The modulation of activity or expression of the JNK and insulinsignaling molecules may be an increase or a decrease. In such an assay,the detection of the activity or expression of the insulin signaling andJNK signaling molecules can be studied using standard techniques. In apreferred embodiment, the agent is identified based on its ability toincrease the activity or expression of the JNK signaling molecule. Inanother preferred embodiment, the agent is identified based on itsability to decrease the activity or expression of the insulin signalingmolecule and the JNK signaling molecule. In yet another embodiment, theagent is identified based on its ability to decrease the activity orexpression of the insulin signaling molecule and to increase theactivity or expression of the JNK signaling molecule.

In preferred embodiments, an agent is screened in an in vitro screeningassays, which may be any of the following: (1) an assay that determineswhether an agent is modulating transcription of a JNK signaling pathwayand insulin signaling pathway polynucleotide; (2) an assay for an agentwhich modulates translation of mRNA or polynucleotides encoding a JNKsignaling pathway molecule and an insuling signaling pathway molecule;(3) an assay for an agent that binds to a JNK signaling pathway and aninsulin signaling pathway polynucleotide or polypeptide; (4) an assayfor an agent that modulates post-translational modification of a JNK orinsulin signaling polypeptide.

For an assay that determines whether an agent modulates transcription ofa JNK or insulin signaling polynucleotide, an in vitro transcription ortranscription/translation system may be used. These systems areavailable commercially, and generally contain a coding sequence as apositive, preferably internal, control. A JNK and/or insulin signalingpolynucleotide is introduced and transcription is allowed to occur.Comparison to transcription products between an in vitro expressionsystems that does not contain any agent (negative control) with an invitro expression system that does contain the agent indicates whether anagent is affecting transcription. Comparison of transcription productsbetween control and the JNK or insulin signaling polynucleotideindicates whether the agent, if acting on this level, is selectivelyaffecting transcription of the JNK or insulin signaling polynucleotide(as opposed to affecting transcription in a general, non-selective orspecific fashion).

For an assay that determines whether an agent modulates translation of aJNK or insulin signaling mRNA or a polynucleotide encoding a JNK orinsulin signaling polypeptide, an in vitro transcription/translationassay as described above may be used, except the translation productsare compared. Comparison of translation products between an in vitroexpression system that does not contain any agent (negative control)with an in vitro expression system that does contain agent indicateswhether an agent is affecting transcription. Comparison of translationproducts between control and the JNK or insuling signalingpolynucleotide indicates whether the agent, if acting on this level, isselectively affecting translation of the JNK or insulin signalingpolynucleotide (as opposed to affecting translation in a general,nonselective or unspecific fashion).

For an assay for an agent that binds to a JNK or insulin signalingpolypeptide, a JNK or insulin signaling polynucleotide is firstrecombinantly expressed in a prokaryotic or eukaryotic expression systemas a native or as a fusion protein in which a JNK or insulin signalingpolypeptide (or fragment thereof) is conjugated with awell-characterized epitope or protein as are well known in the art.Recombinant JNK and/or insulin signaling polypeptide is then purifiedby, for instance, immunoprecipitation using anti-JNK and/or insulinsignaling polypeptide antibodies or anti-epitope antibodies or bybinding to immobilized ligand of the conjugate. An affinity column madeof JNK and/or insulin signaling polypeptide or JNK and/or insulinsignaling polypeptide fusion protein is then used to screen a mixture ofcompounds which have been appropriately labeled. Suitable labelsinclude, but are not limited to flurochromes, radioisotopes, enzymes andchemiluminescent compounds. The unbound and bound compounds can beseparated by washes using various conditions (e.g. high salt, detergent)that are routinely employed by those skilled in the art. Non-specificbinding to the affinity column can be minimized by pre-clearing thecompound mixture using an affinity column containing merely theconjugate or the epitope. A similar method can be used for screening foragents that competes for binding to a JNK and/or insulin signalingpolypeptide. In addition to affinity chromatography, there are othertechniques such as measuring the change of melting temperature or thefluorescence anisotropy of a protein which will change upon bindinganother molecule. For example, a BlAcore assay using a sensor chip(supplied by Pharmacia Biosensor, Stitt et al. (I 995) Cell 80: 661-670)that is covalently coupled to native JNK or insulin signalingpolypeptide or JNK or insulin signaling polypeptide fusion proteins, maybe performed to determine the JNK or insulin signaling polypeptidebinding activity of different agents.

In another embodiment, an in vitro screening assay detects agents thatcompete with another substance (most likely a polypeptide) that binds aJNK or insulin signaling polypeptide. Competitive binding assays areknown in the art and need not be described in detail herein. Briefly,such an assay entails measuring the amount of JNK or insulin signalingpolypeptide complex formed in the presence of increasing amounts of theputative competitor. For these assays, one of the reactants is labeledusing, for example, ³²P.

In another embodiment, an in vitro screening assay detects agents thatmodulate the post-translational modification of a polypeptide. Forexample, techniques can be used for studying phosphorylation of proteins(such as DAF-16) or acetylation of proteins by using antibodies tophospho-epitopes or acetyl group-epitopes.

It is also understood that the in vitro screening methods of thisinvention include structural, or rational, drug design, in which theamino acid sequence, three-dimensional atomic structure or otherproperty (or properties) of a JNK or insulin signaling polynucleotide orpolypeptide provides a basis for designing an agent which is expected tobind to a JNK or insulin signaling polynucleotide or polypeptide.Generally, the design and/or choice of agents in this context isgoverned by several parameters, such as the perceived function of thepolynucleotide or polypeptide target, its three-dimensional structure(if known or surmised), and other aspects of rational drug design.Techniques of combinatorial chemistry can also be used to generatenumerous permutations of candidate agents. For purposes of thisinvention, an agent designed and/or obtained by rational drug designedmay also be tested in the cell-based assays described above.

In a particularly preferred embodiment, the invention further provides amethod for identifying an agent capable of modulating the interactionbetween JNK-1 or a mammalian ortholog thereof and DAF-16 or a mammalianortholog thereof, comprising contacting an assay composition with a testcompound, wherein said assay composition comprises JNK-1 or a mammalianortholog thereof and DAF-16 or a mammalian ortholog thereof, anddetecting an indicator of the interaction between JNK-1 or a mammalianortholog thereof and DAF-16 or a mammalian ortholog thereof, whereinsaid agent is identified based on its ability to modulate theinteraction between JNK-1 or a mammalian ortholog thereof and DAF-16 ora mammalian ortholog thereof.

In a preferred embodiment the interaction is a physical interaction. Inanother preferred embodiment, said interaction is the phosphorylation byJNK-1 or a mammalian orthologue thereof of DAF-16 or a mammalianorthologue thereof.

In one embodiment, the agent inhibits said interaction. In anotherembodiment, the agent promotes said interaction. The invention alsoprovides a novel agent identified according to any of these in vitromethods.

D. Test Compounds

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, K. S. (1997) Anticancer DrugDes. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.)).

In a preferred embodiment, the library is a natural product library,e.g., a library produced by a bacterial, fungal, or yeast culture. Inanother preferred embodiment, the library is a synthetic compoundlibrary.

E. Suitable Controls

Assay methods generally require comparison to a control sample to whichno agent is added. The screening methods described above representprimary screens, designed to detect any agent that may exhibitanti-aging activity. The skilled artisan will recognize that secondarytests will likely be necessary in order to evaluate an agent further.For example, a cytotoxicity assay would be performed as a furthercorroboration that an agent which tested positive in a primary screenwould be suitable for use in living organisms. Any assay forcytotoxicity would be suitable for this purpose, including, for examplethe MTT assay (Promega).

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model, e.g., an animal model to determine theefficacy, toxicity, or side effects of treatment with such an agent.

III. Recombinant Cells and Organisms

The methodologies of the present invention feature cells and organisms,e.g., recombinant cells and organisms, preferably including vectors orgenes (e.g., wild-type and/or mutated genes) as described herein and/orcultured in a manner which results in the overexpression of a JNKsignaling pathway molecule, e.g., JNK, JKK-1, UNC-16, or MEK-1. The term“recombinant” cell or organism includes a cell (e.g., mammalian cell ornematode cell) or organism (e.g., nematode, e.g., C. elegans) which hasbeen genetically altered, modified or engineered (e.g., geneticallyengineered) such that it exhibits an altered, modified or differentgenotype and/or phenotype (e.g., when the genetic modification affectscoding nucleic acid sequences of the cell or organism) as compared tothe naturally-occurring cell or organism from which it was derived.Preferably, a “recombinant” cell or organism of the present inventionhas been genetically engineered such that it overexpresses at least onegene or gene product (e.g., a JNK signaling pathway gene or geneproduct) as described herein. The ordinary skilled will appreciate thata cell or organism expressing or overexpressing a gene product producesor overproduces the gene product as a result of expression oroverexpression of nucleic acid sequences and/or genes encoding the geneproduct.

Suitable host cells and/or whole animals include, but are not limitedto, for example, nematode (e.g., C. elegans), insect, yeast andmammalian cells. In one embodiment, the host cells and/or whole animalsare not insects, e.g., Drosophila.

The term “overexpressed” or “overexpression” includes expression of agene product (e.g., a JNK signaling pathway molecule, e.g., JNK, JKK-1,UNC-16, or MEK-1) at a level greater than that expressed prior tomanipulation of the cell or organism or in a comparable cell or organismwhich has not been manipulated. In particular embodiments of theinvention, overexpression is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 or more foldoverexpression as compared to that expressed prior to manipulation ofthe cell or organism or in a comparable cell or organism which has notbeen manipulated.

In one embodiment, the cell or organism can be genetically manipulated(e.g., genetically engineered) to overexpress a level of gene productgreater than that expressed prior to manipulation of the cell ororganism or in a comparable cell or organism which has not beenmanipulated. Genetic manipulation can include, but is not limited to,altering or modifying regulatory sequences or sites associated withexpression of a particular gene (e.g., by adding strong promoters,inducible promoters or multiple promoters or by removing regulatorysequences such that expression is constitutive), modifying thechromosomal location of a particular gene, altering nucleic acidsequences adjacent to a particular gene such as a ribosome binding siteor transcription terminator, increasing the copy number of a particulargene, modifying proteins (e.g., regulatory proteins, suppressors,enhancers, transcriptional activators and the like) involved intranscription of a particular gene and/or translation of a particulargene product, or any other conventional means of deregulating expressionof a particular gene routine in the art (including but not limited touse of antisense nucleic acid molecules, for example, to blockexpression of repressor proteins).

In another embodiment, the cell or organism can be physically orenvironmentally manipulated to overexpress a level of gene productgreater than that expressed prior to manipulation of the cell ororganism or in a comparable cell or organism which has not beenmanipulated. For example, a cell or organism can be treated with orcultured in the presence of an agent known or suspected to increasetranscription of a particular gene and/or translation of a particulargene product such that transcription and/or translation are enhanced orincreased. Alternatively, a cell or organism can be cultured at atemperature selected to increase transcription of a particular geneand/or translation of a particular gene product such that transcriptionand/or translation are enhanced or increased.

The term “deregulated” or “deregulation” includes the alteration ormodification of at least one gene in a cell or organism that is involvedin a signaling pathway, e.g., the JNK signaling pathway or the insulinsignaling pathway, such that the signal transmission by the pathway isaltered or modified. Preferably, the activity or expression of at leastone enzyme in the pathway is altered or modified such that signaltransmission by the pathway is altered or modified. In a particularembodiment, the methodologies of the present invention featurerecombinant cells or organisms in which the activity or expression of aJNK signaling pathway molecule, e.g., JNK, JKK-1, UNC-16, MEK-1 or amammalian orthologue thereof, is increased. In a preferred embodiment,at least one gene that encodes a JNK signaling pathway molecule, e.g.,JNK, is altered or modified such that the gene product is enhanced orincreased. Other preferred “recombinant” cells or organisms of thepresent invention have a deregulated insulin signaling pathway. Inparticular embodiments, at least one gene that encodes an insulinsignaling pathway molecule, e.g., DAF-2, AAP-1, IRS, AGE-1, PDK-1,AKT-1, AKT-2, or DAF-18 or a mammalian orthologue thereof, is altered ormodified such that the gene product is enhanced or increased. Forexample, in one embodiment, a recombinant cell or organism is designedor engineered such that the activity or expression of a JNK signalingmolecule, e.g., JNK, is increased and the activity or expression of atleast one insulin signaling molecule is decreased, e.g., inhibited. Inanother embodiment, a recombinant cell or organism is designed orengineered such that the activity or expression of a JNK signalingmolecule, e.g., JNK, is decreased and the activity or expression of atleast one insulin signaling molecule is decreased, e.g., inhibited.

IV. Methods of Treatment

The present invention provides methods of treating a subject in needthereof with an agent which modulates JNK signaling, for example, anagent identified according to one of the above-described screeningassays. “Treatment”, or “treating” as used herein, is defined as theapplication or administration of a pharmacological agent of theinvention to a subject, or application or administration of said agentto an isolated tissue or cell line from a subject, in particular anadult subject, an aging subject or an aged subject such that the desiredoutcome is achieved.

The present invention provides a method of enhancing longevity in asubject, involving selecting a subject in need of enhanced longevity,and administering to said subject a pharmacologically effective dose ofan agent that modulates a JNK signaling pathway molecule, whereinmodulation of said JNK signaling pathway molecule in said subjectenhances longevity. In preferred embodiments, the agent increases theactivity or expression of a JNK signaling pathway molecule, e.g., JNK,MKK4, MKK7, JIF scaffold protein, or MAP Kinase Kinase Kinases.Preferably, the agent increases the activity or expression of JNK. In aparticular embodiment of the invention, the method of enhancinglongevity in a subject further involves administering apharmacologically effective dose of an agent that inhibits an insulinsignaling pathway molecule, e.g., insulin receptor, insulin-like growthfactor, insulin receptor substrate, phosphatidylinositol 3-kinase, PTENphosphatase, phosphoinositide kinase 1, protein kinase B and forkheadtranscription factors.

In a preferred embodiment of the invention, a method is provided ofenhancing longevity in a subject, involving selecting a subject in needof enhanced longevity, administering to said subject a pharmacologicallyeffective dose of an agent that upmodulates JNK phophorylation ofDAF-16/FOXO, wherein upmodulation of JNK phosphorylation of DAF16/FOXOin said subject enhances longevity.

The present invention provides a method of preventing or reducingobesity in a subject, involving selecting a subject in need ofpreventing or reducing obesity, administering to said subject apharmacologically effective dose of an agent that modulates JNKsignaling and insulin signaling, wherein modulation of JNK signaling andinsulin signaling in said subject prevents or reduces obesity in saidsubject. In one embodiment of this aspect, JNK signaling and insulinsignaling is modulated by downmodulating the interaction between JNK andDAF-16/FOXO. In another embodiment, JNK signaling and insulin signalingis modulated by downmodulating JNK phosphorylation of DAF-16/FOXO. Invarious embodiments of this aspect, the agent can be, for example, ablocking antibody to DAF-16/FOXO, a blocking antibody to JNK, a form ofDAF-16/FOXO that binds to JNK, a small molecule that inhibits JNK, anagent that modulates the interaction of JNK and DAF-16/FOXO such thatDAF-16/FOXO nuclear translocation is modulated.

The present invention further provides a method of preventing ortreating type II diabetes in a subject, involving selecting a subject inneed of prevention or treatment for type II diabetes, administering tosaid subject a pharmacologically effective dose of an agent thatmodulates JNK signaling and insulin signaling, wherein modulation of JNKsignaling and insulin signaling in said subject prevents or treats typeII diabetes in said subject. In one embodiment, the JNK signaling andinsulin signaling is modulated by down modulating the interactionbetween JNK and DAF-16/FOXO. In another embodiment, JNK signaling andinsulin signaling is modulated by downmodulating JNK phosphorylation ofDAF-16/FOXO. In various embodiments, the agent can be, for example, ablocking antibody to DAF-16/FOXO, a blocking antibody to JNK, a form ofDAF-16/FOXO that binds to JNK, a small molecule that inhibits JNK, anagent that modulates the interaction of JNK and DAF-16/FOXO such thatDAF-16/FOXO nuclear translocation is modulated.

Such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the target genemolecules of the present invention or target gene modulators accordingto that individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

The modulators of the present invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, protein,antibody, or modulatory compound and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation),transdermal (topical), and transmucosal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit large therapeutic indices are preferred. Althoughcompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the EC50 (i.e., the concentration ofthe test compound which achieves a half-maximal response) as determinedin cell culture. Such information can be used to more accuratelydetermine useful doses in humans. Levels in plasma may be measured, forexample, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES Example 1 Combined Reduction of Function Mutations in a JNKPathway Gene and daf-2 Leads to an Enhancement (jnk-1, unc-16, jkk-1) orSuppression (mek-1) of Life Span Extension Associated with daf-2 Mutant

To identify other signaling pathways downstream of DAF-2, animals weregenerated having a reduction of function mutation in the DAF-2 receptorcombined with a mutation in a kinase whose signaling pathway isimplicated in mammalian insulin signaling. It was postulated that sincemutations in daf-2 affect so many pathways, a novel interaction could beidentified by looking at a subset of the phenotypes displayed in thesedouble mutants. Such genes might not yet have been found because theyonly affect a subset of the daf-2 mutant phenotypes. Alternatively, suchdouble mutants in combination with daf-2 could be dead. It has beensuggested that the complete loss of function mutation in daf-2 is lethal(Gems D. et al. (1998) Genetics 150: 129-155), and a knock-out of theinsulin receptor in mice creates pups that survive only a few weeks(Sone H. et al. (2001) Trends in Mol Med 7:320-322).

This approach was applied to the JNK signaling pathway. First, a panelof C. elegans mutants were produced. C. elegans strains were obtainedcontaining a reduction-of-function mutation in jkk-1, mek-1, jnk-1 orunc-16. C. elegans strains were also constructed containing a loss orreduction-of-function mutation in jkk-1, mek-1, jnk-1 or unc-16 incombination with a reduction-of-function mutation in daf-2 or age-1. Thephenotypes of these mutants were assessed using a standard assay forlife span in order to test whether these JNK pathway genes modulated thelife span extension associated with a single daf-2 or age-1 mutant.

Strains and Media

Strains included: N2 (wild type), jnk-1 (gk7), jkk-1 (km-2), mek-1(ks54), unc-16 (e109), age-1 (hx546), daf-2 (e1370), daf-16 (mu86).Strains were obtained from the Center of Caenorhabditis elegans GeneticsCenter (University of Minnesota, Minneapolis, Minn.). Nematodes werecultured under standard conditions (Brenner S. (1974) Genetics77:71-94).

Strain Construction

To construct double mutant strains, the following general approach wasused: daf-2, age-1, or daf-16 males were obtained by heat-shock at 30°C. for 6 hours, and these males were used to mate with jnk-1, jkk-1,mek-1, or unc-16 hermaphrodites. For example, daf-2 (e1370) males weremated to jnk-1 hermaphrodites at 15 or 20° C., and 5-7 (15) or 3-4 (20)days later, putative cross progeny were singled to individual plates at25° C. and allowed to have progeny. Three days later, the plates werescored for the presence of dauers (daf-2). Plates that segrated dauerswere kept. Dauers were then returned to 15° C. to recover and singled toindividual plates. These recovered dauers were allowed to have progenyand then were tested for presence or absence of the jnk-1 mutation byPCR. Matings were done typically at 15° C. and 20° C. depending on thestrain.

Strain characterization The identity of all single and double mutantswere confirmed by PCR as well as by phenotypic and complementationanalysis. Homozygote F2 worms were identified by dauer formation at 25°C. (daf-2) or 27° C. (age-1), phenotype (unc-16 and mek-1), and PCRamplification (jnk-1, jkk-1, and daf-16). The condition for PCR was 34cycles of 94° C. for 30 sec, 50° C. for 1 min, and 72° C. for 3 min,followed by 72° C. extension for 10 min. Primers used for PCR were jnk-1(5′-ACAGTGGAACAGGAGGAGGA-3′ (SEQ ID NO:1) and 5′-ATGCCTATCTGCCTGAGAGC-3′(SEQ ID NO:2), jkk-1 (5′-AGGAGAAAAGCAAGTTGTCG-3′ (SEQ ID NO:3) and5′-GCAGCAGCTTTCACAACAC-3′ (SEQ ID NO:4), and daf-16(5′CAATGAGCAATGTGGACAGC-3′ (SEQ ID NO:5) and 5′-CCGTCTGGTCGTTGTCTTTT-3′(SEQ ID NO:6)).

Life Span Assay

Life span assays were performed as described (Apfeld & Kenyon (1998)Cell 95:199-210). Briefly, life span was determined on seeded NGM(nematode growth media) plates at 20° C. Adult hermaphrodites werepicked (4-10 per plate) from each strain and allowed to undergo one fullgeneration at 15° C. or 20° C. From these plates, individual L4s oryoung adults were picked to plates at 20° C. containing 400 μg/ml FUDR.FUDR blocks DNA synthesis and causes animals to lay eggs that do notdevelop, thereby eliminating the need to transfer animals throughout thelife span assay. Survival of the hermaphrodites was measured every fewdays by tapping. Animals were considered dead if no pharyngeal pumpingwas evident and they failed to respond to repeated prodding (Johnson T.et al. (1982) PNAS 79:6603-7).

Single Mutant Phenotypes

Several single mutants in the JNK pathway were already known to bestress sensitive. Many studies have shown a correlation between stressresistance and extended life span. The life span of single reduction offunction mutants of mek-1, jkk-1 and jnk-1 and unc-16 were thereforeexamined to determine if these mutations affected life span whencompared to wild type or to the previously characterized reduction offunction mutant of daf-2 (e1370).

Results are shown in FIGS. 3 a-d. The daf-2 mutant (e1370), aspreviously shown, significantly extended life span relative to the N2control strain. Individual reduction of function mutations in the JNKpathway genes, including unc-16 and mek-1, showed no effect on life spanrelative to the N2 control. Both the jnk-1 and jkk-1 mutants showed aslight, but statistically significant, decrease in life span. The meanlife span of the strains were: wild-type=17.1±3.82 (n=45),jnk-l(gk7)=13.1±3.5 (n=50), jkk-l(km2)=13.6±2.18 (n=50), mek-1=17.9±4.52(n=49), unc-16=15.6±3.7 (46). These data are the mean±standarddeviation, (n)=total number of animals tested.)

The demonstration that a reduction of function mutation in jnk-1uniquely caused a decrease in mean life span was intriguing, given thatjnk-1 and mek-1 mutants are hypersensitive to heavy metals (VillanuevaA. et al. (2001) EMBO J 20:5114-5128). These data demonstrated that thecorrelation between resistance to heavy metal stress and the long-livedphenotype was not strictly true. Alternatively, there may be aredundancy among these genes for life span, and the mkk-4 gene wascontributing an effect.

Double Mutant Phenotypes

The introduction of a reduction of function mutation in either jnk-1,unc-16 or jkk-1, in combination with a reduction of function mutation inthe insulin-like receptor gene daf-2, led to a striking, synergisticenhancement of the life span extension that was associated with thesingle daf-2 mutant. The mean and maximum life span of double mutantstrains were: daf-2;jnk-1 (48.3, 71), daf-2;jkk-1 (47.5, 71), and daf-2,unc-16 (60.9, 100) (mean life span, maximum life span). Thedemonstration that mutations in jkk-1 or unc-16, which do not affectlife span on their own, showed a striking synergistic effect when placedin combination with another mutation that caused a change in signalingin the insulin pathway (and therefore the activity of DAF-16), was noveland unexpected. This result clearly demonstrated a regulatory role forjnk-1, unc-16 and jkk-1 in aging, and indicated that, in the absence ofdaf-2, the JNK pathway genes jnk-1, unc-16 and jkk-1 function asnegative modulators of lifespan in C. elegans.

The introduction of a reduction of function mutation in mek-1 incombination with a reduction of function mutation in daf-2 (e1370) ledto a partial suppression of the life span extension associated with thesingle daf-2 mutant. (daf;mek-1 (25.4, 51) (mean life span, maximum lifespan)). This result contrasted with that obtained with mutants of theother JNK pathway genes, and showed that mek-1, in the absence of daf-2,acts as a positive modulator of life span.

Example 2 Combined Reduction of Function Mutations in a JNK Pathway Gene(jnk-1, unc-16, jkk-1 or mek-1) and age-1 Synergistically Enhances LifeSpan Extension of age-1 Mutant

C. elegans strains were constructed harboring reduction of functionmutations in age-1 in combination with a reduction of function mutationin either jnk-1, unc-16, jkk-1 or mek-1. The phenotypes of these mutantswere assessed using a standard assay for life span, as described in theMaterials and Methods of Example 1, in order to determine whether theseJNK pathway genes modulated the life span extension associated with asingle age-1 mutant.

Results are shown in FIG. 4. The reduction of function mutation in age-1significantly extended life span relative to an N2 control strain, aspreviously described. Single mutants for JNK pathway genes, includingunc-16, jkk-1 and mek-1, showed no effect on life span, while the jnk-1mutant showed a slight, but statistically significant, decrease in lifespan relative to the N2 control.

The introduction of a reduction of function mutation in jnk-1, unc-16,jkk-1 or mek-1 in combination with a reduction of function mutation inage-1 led to a synergistic enhancement of the life span extensionassociated with the age-1 single mutant: age-1;jnk-1 (28.9, 48),age-1;jkk-1 (33.7, 48), age-1;mek-1 (30.4, 44), and age-1;unc-16 (22.3,53) (mean life span, maximum life span). These data further demonstrateda regulatory role for the JNK pathway in aging, and indicate that, inthe absence of age-1, each of the JNK pathway genes examined functionsas a negative modulator of life span.

Example 3 Enhancement of Life Span Extension in a daf-2/jnk-1 DoubleMutant Requires daf-16

To test whether the synergistically enhanced life span extensionassociated with the double mutant daf-2/jnk-1 was dependent on daf-16, aC. elegans strain was constructed that had a null mutation in daf-16 incombination with the reduction of function mutations in jnk-1 and daf-2.The phenotype of this mutant was assessed using a standard assay forlife span, as described in the Materials and Methods of Example 1. Lifespans were examined to determine whether a mutation in daf-16 modulatedthe synergistic life span extension associated with the daf-2/jnk-1mutant.

Results are shown in FIG. 3. The daf-16 mutant showed a slight reductionin life span relative to an N2 control strain, consistent with previousreports. As demonstrated in Example 1, a single jnk-1 mutant exhibited aslight decrease in life span relative to the N2 control, and the doublejnk-1/daf-2 mutant showed a synergistic life span extension comparedwith that of the daf-2 single mutant. Importantly, the introduction of athird mutation in the insulin-signaling gene, daf-16, completelysuppressed the synergistic life span extension phenotype of jnk-1/daf-2to that of the N2 control (daf-16;daf-2;jnk-1(12.1, 20) (mean life span,maximum life span)). This result demonstrated that jnk-1, like daf-2,requires daf-16 for its life span regulating effects. Taken together,these results placed jnk-1 acting upstream of daf-16 in C. elegans.

Example 4 Evaluation of Stress Resistance and Body Movement Coordinationin Mutants of Genes in the JNK Pathway (jnk-1, unc-16, jkk-1, mek-1) inCombination with daf-2 or age-1

Reduction of function mutations in jnk-I, jkk-1 and mek-1 result indefects in coordinated body movements and/or resistance to stress, suchas heavy metals (Villanueva A. et al. (2001) EMBO J 20:5114-5128; KogaM. et al. (2000) EMBO J 19:5148-5156; Kawasaki M. et al. (1999) EMBO J18:3604-3615). These defects in coordination are primarily due to thefact that at least jnk-1 and jkk-1 are expressed in both the cell bodiesand the axons of most neurons. In order to evaluate resistance tovarious stresses, including UV, oxidative and heat stress, as well asbody movement coordination, C. elegans strains harboring a reduction offunction mutation in daf-2 or age-1 in combination with a reduction offunction mutation in jnk-1, jkk-1, mek-1, unc-16 or mkk-4 are generatedas in Examples 1 and 2. Mutants are then examined for stress resistanceand the movement phenotype as follows:

UV stress Approximately 30-40 L4˜young adult animals are removed from aseeded plate, washed in 1×S-Basal, then transferred to an unseeded NGMplate. Animals are exposed to 40 J/m2 in a Stratalinker 2400(Stratagene). Animals are removed from the unseeded plate and placed ona seeded one. Life span is calculated from the day of UV treatment. UVtreatment often leads to egg laying defects and bagged adults. Theseanimals are censored from life span calculations.

Oxidative Stress

For each strain to be tested, 100 L1 animals are placed to develop toadulthood on NGM plates containing different concentrations of paraquat(0 mM, 0.2 mM, 0.4 mM, 0.6 mM, and 0.8 mM) (Feng et al. (2001) Dev.Cell. 1:1-20). For each strain, worms are monitored each day until 6days after the first worms become adults. The percentage of worms thatreach adulthood is expressed as survival.

Heat Stress

Intrinsic thermotolerance is measured as a percent of a cohort ofL4˜young adult worms that survive a near-lethal heat shock.Specifically, 30-40 wild type or mutant adults are placed on a seededNGM plate and left to lay eggs for 3-4 hours. Adults are removed andeggs are allowed to develop until 3 days past L4 molt. Next, 30-40rolling adults are placed on a small seeded NGM plate at 35° C. for 24hours. The worms are then scored for viability.

Body Movement Coordination Assay

Single L4˜young adult worm is placed on a seeded NGM plate and bodybending per minute is recorded by manual counting under the microscope.The locomotion of worms is observed after 1 min, 10 min, and 60 min bydrawing the line on the plate lid along the tracks made by worms.

Example 5 Materials and Methods for Examples 6-15

Construction of C. elegans Strain Overexpressing jnk-1

The jnk-1 genomic sequence, including the promoter region, 3 kb of DNAupstream of the start codon, the entire coding region and 500 bp of the3′-UTR, was amplified by PCR of N2 genomic DNA (upstream primer5′-GCGTCCTCCTGTGCTCACTC (SEQ ID NO:7), and downstream primer5′-CCCACGACAACTGCTACAAC (SEQ ID NO:8)) After gel purification, a 9.3 kbfragment was injected into N2 animals at 50 ng/μl along with aco-injection marker, pRF4 rol-6, at 100 ng/μl. A stable transgenic linewas generated by irradiating with UV at 300 J/m² in order to integrateextrachromosomal arrays into the chromosome. Several extrachromosomaland integrated lines were established and compared for each experiment.

Strain Construction

To construct double mutant strains overexpressing jnk-1 and containingeither a reduction of function mutation in jkk-1, mek-1 or unc-16, orthat expressed GFP-tagged DAF-16, the following general approach wasused: jnk-1 overexpression hermaphrodites were crossed with males ofjkk-1, mek-1, unc-16, or daf-16::GFP, which were obtained by heat shockat 30° C. or which occurred naturally. For individual F2 cross progeny,jnk-1 overexpression was confirmed by roller phenotype or by PCR(upstream primer 5′-ACAGTGGAACAGGAGGAGG (SEQ ID NO:9), and downstreamprimer 5′-ATGCCTATCTGCCTGAGAGC (SEQ ID NO:10)), jkk-1 deletion wasconfirmed by single worm PCR (upstream primer 5′-AGGAGAAAAGCAAGTTGTCG(SEQ ID NO:11), and downstream primer 5′-GCAGCAGCTTCTCACAACAC (SEQ IDNO:12)), mek-1 was confirmed by hypersensitivity to CuSO₄, unc-16 wasconfirmed by unc phenotype, and daf-16::GFP was characterized by GFPsignal. A daf-2:daf-16::GFP double mutant was made by crossing daf-2males with daf-16::GFP hermaphrodites. Approximately 20 putative F1roller cross progeny were transferred to 25° C. Homozygote daf-2 rollerswere selected by dauer phenotype at 25° C. Each double mutant wasconfirmed again using F3 progeny. More detailed protocols for individualstrains are as follows:

For jkk-1;lpIn2 and mek-1;lpIn2: jkk-1 and mek-1 males were obtained byheat shock at 30° C. for 6 hr or spontaneously on the plate and mated tothe lpIn2 hermaphrodites. From the mating plate, twenty putative F1cross progeny were picked to individual plates and allowed to haveprogeny. From at least 2 individual F1 plates that segregated bothrollers and non-rollers (indicating cross progeny), 20-30 F2 rollerswere picked to individual plates and allowed to have progeny. Once theparents had progeny (F3), the F2 parents were tested forhypersensitivity to CuSO4 for mek-1 or for the presence of the jkk-1deletion mutation (5′-AGGAGAAAAGCAAGTTGTCG, 3′-GCAGCAGCTTCTCACAACAC) andjnk-1 overexpression (5′-ACAGTGGAACAGGAGGAGG, 3′-ATGCCTATCTGCCTGAGAGC)by PCR. Then, the plates that segregated 100% rollers (F3) were kept toestablish the strain. The crosses were done at 20° C.For lpIn2;daf-16::gfp and jkk-1;lpIn2;daf-16::gfp: Spontaneouslyobtained daf-16::gfp males were mated to either lpIn2 or jkk-1;lpIn2hermaphrodites. The crosses were done as described above except that F2worms were screened for both GFP and the roller phenotype.For daf-2;lpIn1: daf-2(e1370) males were mated to lpIn1 hermaphrodites.About 20 putative F1 roller cross progeny were transferred to 25° C. andallowed to have F2 progeny. From plates segregating rollers andnon-rollers, wild type and dauers, 15-20 rolling dauers were picked toindividual plates and allowed to recover at 15° C. The plates werescored for 100% roller progeny in F3. The rollers were then retested fordauer formation at 25° C.Transgenic Worms

jnk-1 genomic DNA including 3 kb of the promoter region, the entirecoding region, and 500 bp 3′-UTR was amplified from N2 genomic DNA byPCR (5′-GCGTCCTCCTGTGCTCACTC, 3′-CCCACGACAACTGCTACAAC). After gelextraction (Qiagen), the 9.3 kb fragment was injected at 50 ng/μl intothe gonad of N2 worms along with pRF4, (rol-6(su1066) plasmid) as aco-injection marker (100 ng/μl) (14) to generate stable extrachromosomaltransgenic lines. Two independent integrated lines (lpIn1 and lpIn2)were generated from one extrachromosomal line (lpEx1). Severalextrachromosomal and integrated lines using at least two differentmarkers were established and showed similar results.

Life Span Analysis

Life span assays were performed at 20° C. Adult hermaphrodites from eachstrain were transferred to fresh nematode growth medium (NGM) plates at20° C. and allowed to undergo one full generation to ensure the wormswere well fed and had not gone through dauer. L4s or young adults werethen transferred to new NGM plates containing 0.1 mg/ml of5′flourodeoxyuridine (FUDR) to prevent the growth of progeny (15).Animals were tapped every 2-3 days and scored as dead when they did notrespond to the platinum wire pick. We determined survival from the pointwhen the worms were transferred to the FUDR plate and therefore lifespan is defined as the days the worms survived (day 1). All the lifespan assays were repeated at least three times. For life span analysison RNAi plates, a single colony from each RNAi clone was grown in LBbroth containing 50 μg/ml ampicillin and 12.5 μg/ml tetracycline to OD0.5-1.0. The bacteria were then seeded onto an NGM plate containing 1 mMisopropylthiogalactoside (IPTG), 50 μg/ml of ampicillin, and 0.1 mg/mlFUDR. Seeded plates were dried overnight at room temperature and thenstored at 4° C. for subsequent use.

Visualization of DAF-16 Translocation

DAF-16 translocation was visualized by a GFP microscope (Zeiss, Axioskop2 plus) equipped with a Hamamtsu C4742-95 digital camera. Images wereobtained using OpenLab 3.1.4 software (Improvision).

Oxidative Stress Assay

For oxidative stress assays, 30˜40 young adults were transferred to96-well plates (5˜6 worms/well) containing 40 μl of 150 mM paraquat.Worms were scored for survival every 30 minutes by tapping them with aplatinum wire pick. The oxidative stress assay was repeated at least 5times.

Plasmid Construction and Transfection

Full-length daf-16 a1 and jnk-1α cDNAs were amplified by RT-PCR(Invitrogen) using total RNA isolated from N2 worms (Ambion). The cDNAswere cloned into the mammalian expression vector with either Flag-tag(p3XFLAG-myc-CMVTM-26, Sigma) or Xpress-tag (pcDNA3.1B, Invitrogen) toobtain Flag-tagged DAF-16 and Xpresstagged JNK-1. Plasmids weretransfected into COS-7 cells and harvested after 48 hours. Fordetermining whether DAF-16 could serve as a substrate for JNK-1, N- andCterminal portion of DAF-16 were amplified by PCR using N2 total RNA andcloned into pET-24b vector (Novagen) between HindIII and XhoI. TheHis-tagged DAF-16 fusion protein was expressed in E. coli (BL-21) andpurified under native condition with Ni-NTA His-Bind resin (Novagen).

Antibody Production

A C-terminal portion of JNK-1α (228-451) was cloned into a His-taggedexpression vector (pET-24b, Novagen), expressed in bacteria (BL21(DE3))and purified using Ni-NTA His-bind resin (Novagen). Polyclonal antiserumwas raised against the recombinant protein in rabbit (Capralogics Inc.).

Immunoblotting, Immunoprecipitation and Kinase Assay

For phospho-JNK immunoblotting, worms were grown on 10 cm NGM plates andground with stainless steel Dounce homogenizer in lysis buffer (20 mMTris (pH 7.4), 137 mM NaCl, 2 mM EDTA, 10% Glycerol, 1% Triton X-100, 25mM β glycerophosphate, 1 mM NaVO3, 1 mM PMSF, 10 ug/ml Leupeptin, 10ug/ml

Aprotinin). Proteins were separated by SDS-PAGE and immunoblotted withphospho-JNK antibody (Cell signaling) or JNK-1 antibody (raised againstC. elegans JNK-1). For immunoprecipitation, COS-7 cells were lysed inthe same lysis buffer and following the centrifugation at 14,000 g for10 min, the supernatant was pre-cleared with 50 μl of proteinG-Sepharose bead (Amersham). This was then incubated with anti-Xpressantibody (Sigma) along with fresh bead O/N at 4° C. After severalwashes, lysates were boiled with sample buffer. Proteins were separatedby SDS-PAGE and immunoblotted with anti-Flag antibody (Invitrogen). Forkinase assay, Protein G-Sepharose (Pharmacia-LKB Biotechnology) beadswere incubated for 3 to 4 h at 4° C. with anti-Xpress antibody, washedtwice with the lysis buffer as described above, and then incubated withlystates of COS-7 cells transfected with Xpress-JNK-1 O/N at 4° C.Complexes were washed three times with the lysis buffer and once withkinase buffer (25 mM HEPES (pH 7.4), 25 mM β-glycerophosphate, 25 mMMgCl2, 0.1 mM NaVO3, 2 mM DTT). The kinase activity of JNK-1 wasmeasured by adding 20 μl of kinase buffer containing 50 μM [γ-32P]ATP(10 Ci/mmol) and 1 μg of N- or C-terminal portion of DAF-16 or GST-c-Jun(1-79) and incubating at 30° C. for 30 min. The reactions wereterminated by boiling in sample buffer. Proteins were resolved bySDS-PAGE and analyzed by autoradiography.Heat Resistance Assay and DAF-16 Translocation Assay

To measure heat stress resistance, 50 young adult worms were picked ontoNGM plates and kept at 35° C. Animals were tapped every hour and scoredas dead when they did not respond to the platinum wire pick. Assays wererepeated at least 3 times. For DAF-16 translocation assay, 10˜15 L4swere placed on NGM plates at 35° C. After 30 minutes, worms wereimmediately mounted onto the slide with 0.1% of sodium azide in S-basalbuffer. We visualised the nuclear translocation of DAF-16 was visualizedwith a fluorescence microscope (Zeiss) equipped with a Hamamatsu digitalcamera and analysed by Openlab software. We scored each animal as havingcytosolic localization (Cyt); nuclear localization (nuc) whenlocalization is observed throughout the entire body from head to tail,or Intermediate (Int) when there is a visible nuclear localization butnot as complete as Nuc. The number of worms with each level of nucleartranslocation was counted. The translocation assay was repeated at least5 times by two different individuals.

PCR Primers Used for Cloning

Flag-DAF-16: 5′-GAAGATCTGGAGATGCTGGTAGATCAGGG,3′-CGGGGTACCTTACAAATCAAAATGAATAT; Xpress-JNK-1:5′-CGGGATCCGGAGGAACGATTATCCACAAC, 3′-GCTCTAGATCAGGAATAAATGTCATGGG;Xpress-JNK-1 (APF): 5′-GAGGCATTCATGATGGCTCCTTTCGTTGTGACAAGATAC,3′-GTATCTTGTCACAACGAAAGGAGCCATCATGAATGCCTC; His-DAF-16 (Nterminalfragment): 5′-CCCAAGCTTGGCCTATACGGGAGCAATGAGC,3′-CCGCTCGAGCGGACGGAAAGATGATGGAACG; His-DAF-16 (C-terminal fragment):5′-CCCAAGCTTGGCGGAGCCAAGAAGAGGATA, 3′-CCGCTCGAGCGCAATTGGAAGAGCCGATGAA

Example 6 Overexpression of jnk-1 Extends Lifespan

Single Reduction-of-Function Mutant Phenotypes

In similar experiments to those described in Example 1, the life span ofsingle reduction of function mutants of mek-1, jkk-1, jnk-1 and unc-16were examined for the effects of these mutations on life span ascompared to wild type. The strains, construction and characterizationthereof, and life span analysis were as set forth in Example 1. Results,as shown in FIG. 6, demonstrate that mek-1 and unc-16 mutant showedlittle or no effect on life span, while the jnk-1 and jkk-1 mutantsshowed a statistically significant decrease in life span. The mean lifespan of the strains were: wild-type=16.7±0.18, mek-1=16.5±0.38,unc-16=15.7±0.31, jnk-l(gk7)=14.1±0.22, jkk-l(km2)=13.8±0.20. These dataare the mean±standard error. These results are consistent with thosefound in Example 1.

Overexpression of jnk-1 Extends Lifespan

A 9.3 kb region of genomic DNA spanning the jnk-1 gene, including 3 kbof the promoter region and 500 bp of the 3′-UTR (depicted in FIG. 7A),was amplified by PCR. This DNA was then injected into the gonads of wildtype worms (N2) along with the coinjection marker, rol-6, as describedabove. By using this approach, a jnk-1 overexression transgenic line wascreated that contained extra copies of the jnk-1 gene, as confirmed bysingle-worm PCR analysis (FIG. 7B). The expression level of JNK-1 inthis transgenic line was examined using RT-PCR and the results arepresented in FIG. 7C. These results demonstrated that JNK-1 expressionwas elevated by approximately 10-fold.

The phenotype of the jnk-1 overexpression strain was assessed using astandard assay for life span to test whether overexpression of jnk-1modulates life span. As shown in FIG. 8, overexpression of jnk-1increased life span significantly. The mean life span of the strainswere: wild-type=16.7±0.18, jnk-1 overexpression=22.4±0.45. These dataare the mean±standard error. Taken together with results from thereduction of function mutants, these results indicated that the JNKsignaling pathway is required to maintain the normal lifespan (asdemonstrated in the present Example and in Example 1) while additionalJNK signaling can extend lifespan. These results suggested that the JNKsignaling pathway regulates lifespan in a dose-dependent manner in C.elegans.

Example 7 DAF-16 is Required for Lifespan Extension by jnk-1Overexpression and is Localized to the Nucleus in a jnk-1 OverexpressionStrain

The next question addressed was whether daf-16 was required for theobserved lifespan extension upon overexpression of jnk-1. To addressthis question, the lifespan of the jnk-1 overexpression strain wasmeasured on a daf-16 RNAi plate or on an empty RNAi vector plate ascontrol (L4440) (as described in Nature Genetics 2003, 33:40-48). Theresults of this analysis, which are presented in FIG. 9, revealed thatthe lifespan extension associated with jnk-1 overexpression wascompletely suppressed by daf-16 RNAi. These results indicated thatdaf-16 is required for the lifespan extension associated with jnk-1overexpression.

The translocation of DAF-16 from the cytoplasm into the nucleus is a keyevent in lifespan regulation. Given that the lifespan extensionexhibited upon jnk-1 overexpression required daf-16, it was possiblethat jnk-1 overexpression mediated its effect on lifespan by modulatingDAF-16 localization. To examine localization of DAF-16 in the jnk-1overexpression strain, a jnk-1 overexpression worm was crossed with adaf-16::GFP strain, and DAF-16 translocation was visualized using a GFPmicroscope. The results of this experiment are presented in FIGS. 10A-C.In the wild-type strain, DAF-16 was localized predominantly in thecytosol (FIG. 10A), while in the daf-2 mutant strain DAF-16 wastranslocated to the nucleus (FIG. 10B), as previously described.Strikingly, DAF-16 was similarly localized to the nucleus in a jnk-1overexpression strain (FIG. 10C). These results revealed a novelmechanism by which translocation of DAF-16 is regulated, and suggestedthat jnk-1 overexpression extends lifespan by localizing DAF-16 into thenucleus.

Example 8 Upstream Kinases jkk-1 and mek-1, but not unc-16, are Requiredfor Life Span Extension by jnk-1 Overexpression

The next question addressed was whether the kinases that act upstream ofjnk-1, including jkk-1, mek-1, or unc-16, are required for the observedlifespan extension upon overexpression of jnk-1. To address thisquestion, a jnk-1 overexpression worm was crossed with C. elegansreduction of function mutants for other components in JNK signalingpathway, including jkk-1, mek-1, and unc-16, and the life span of thesestrains were analyzed. Results of these experiments are presented inFIGS. 11A-C. Lifespan extension by jnk-1 overexpression was completelysuppressed by a reduction of function mutation in jkk-1 (FIG. 11A). Themean life span of the strains were: wild-type=16.7±0.18,jkk-l(km2)=13.8±0.20, jnk-1 overexpression=18.8±0.48, jkk-1;jnk-1overexpression=14.8±0.38 (mean±standard error). Similarly, lifespanextension by jnk-1 overexpression was completely suppressed by areduction of function mutation in mek-1 (FIG. 11B). The mean life spanof the strains were: wild-type=16.7±0.18, mek-l(ks54)=16.5±0.38, jnk-1overexpression=18.8±0.48, mek-1;jnk-1 overexpression=15.2±0.50(mean±standard error). In contrast, life span extension by jnk-1overexpression was not affected by a mutation in unc-16 (FIG. 11C). Themean life span of the strains were: wild-type=16.7±0.18,unc-16(e109)=15.7±0.31, jnk-1 overexpression=21.0±0.55, unc-16;jnk-1overexpression=19.6±0.30 (mean±standard error). These results indicatethat jkk-1 and mek-1, but not unc-16, are required for lifespanextension by jnk-1 overexpression.

Example 9 Overexpression of jnk-1 in C. elegans Confers Resistant toStress

A jnk-1 overexpression worm was challenged with 150 mM of paraquat orexposed to heat shock at 35° C., and the worms were then scored forsurvival every 30 minutes or 1 hour, respectively. The results of theseexperiments, as presented in FIGS. 12A-B, demonstrate that a jnk-1overexpression C. elegans strain was significantly resistant to bothheavy metal stress (FIG. 12A) and to heat shock stress (FIG. 12B). Theseresults suggest that lifespan extension by jnk-1 overexpression is dueto stress resistance.

Example 10 DAF-16 is Required for Lifespan Extension by jnk-1Overexpression

Experiments were next carried out to confirm the results obtained withthe jnk-1 overexpressing worms obtained in Example 6 above. jnk-1overexpressing worms were created essentially as described above inExample 6. Briefly, 9.3 kb of jnk-1 genomic DNA was amplified including3 kb of the promoter region, the entire coding region, and 500 bp of the3′-UTR by PCR. The entire PCR fragment was injected into the gonad ofwild-type worms along with the co-injection marker, rol-6(su1066) (14),to create jnk-1 overexpression transgenic worms. From one of severalextrachromosomal lines (lpEx1), two independent integrated lines (lpIn1,lpIn2) were derived. Both lpIn1 and lpIn2 exhibit similar increases inexpression of the jnk-1 transcript, as determined by RT-PCR (FIG. 14).The transcription level was measured in two independent integratedtransgenic lines by RT-PCR (5′-ACAGTGGAACAGGAGGAGG,3′-ATACGGAAGTGGAGGTGGAG) and compared to the wild-type. Data for eachstrain (% of N2): wild-type (N2), 100%; jnk-1, 0%; lpIn1, 1368%; lpIn2,1432%. lpIn1 and lpIn2 extend life span by 40% compared to the control,implying that jnk-1 is a positive regulator of life span (N2+pRF4(rol-6(su1066) plasmid) control: 15.2±0.3 days, lpIn1: 20.9±0.6 days(P<0.0001), and lpIn2: 18.8±0.5 (P<0.0001); FIG. 15).

In C. elegans, daf-16 plays a central role in life span regulation suchthat a mutation in daf-16 suppresses the life span extension of daf-2,age-1 or other long-lived mutants (20-23). Therefore, the integratedline lpIn1 was used in experiments similar to those described above inExample 7 to determine whether life span extension by jnk-1overexpression is also dependent on daf-16. Life span analysis of jnk-1overexpression worms on daf-16 RNAi was carried out as described inExample 7. In agreement with previous work (24), daf-16 RNAi shortenslife span (wild type on control RNAi: 17.6±0.4 days, on daf-16 RNAi:14.4±0.4 days; FIG. 16A). Life span extension by jnk-1 overexpression iscompletely suppressed by daf-16 RNAi (lpIn1 on control RNAi: 19.1±0.6days, on daf-16 RNAi: 14.7±0.4 days; FIG. 16A). These results confirmthose described in Example 7 and indicate that daf-16 is required forlife span extension by jnk-1 overexpression.

Example 11 The jnk-1 Pathway and Insulin-Like Pathway Regulate Lifespanin Parallel and the Two Pathways Converge onto daf-16

If jnk-1 exerts its effect through the insulin-like pathway, jnk-1overexpression would not have an additional effect on life span in thebackground of mutations in the insulin-like pathway. However, ifcombining a hypomorphic mutation in the insulin-like pathway with jnk-1overexpression produces a further increase in life span, then this wouldbe consistent with the possibility that jnk-1 acts in a parallelpathway. To address this question, a jnk-1 overexpression line (lpIn1)was crossed with daf-2(e1370) and the combination was found tosignificantly extend life span beyond daf-2(e1370) (daf-2: 44.0±0.7days, daf-2;lpIn1: 53.3±1.7 days (P<0.0001); FIG. 16B). Furtherdownstream of daf-2 in the insulin-like signaling pathway are thekinases AKT-1 and AKT-2 (25). Since experiments in mammalian system haveshown an interaction between the AKT and JNK pathways (8, 9), the lifespan of lpIn1 in combination with akt-1/akt-2 was examined. A doublemutant strain containing null mutations (26) in both akt-1(ok525) andakt-2(ok393) was generated. However, 100% of the akt-1(ok525);akt-2(ok393) double mutants arrest at dauer larval stage at alltemperatures. To circumvent this problem, akt-1(ok525); akt-2(ok393)double mutants and the akt-1(ok525); akt-2(ok393); lpIn1 strain weregrown on daf-16 RNAi to bypass the dauer larval stage and life span wasthen tested on regular plates. The akt-1(ok525); akt-2(ok393); lpIn1strain shows a life span extension beyond the akt-1(ok525); akt-2(ok393)double mutant alone (wild-type (N2): 14.9±0.4 days, akt-1(ok525);akt-2(ok393): 34.2±0.8 days, akt-1(ok525); akt-2(ok393); lpIn1: 38.8±0.9days; FIG. 16C). Similar results were obtained with akt-1/2 RNAi (FIG.17). In addition, life span extension by either daf-2 mutation alone orin combination with jnk-1 overexpression (lpIn1) was found to becompletely suppressed by daf-16 (1,3, 4)). These results suggest thatjnk-1 regulates life span in parallel to the insulin-like pathway butboth converge onto daf-16. Alternatively, it is also possible that jnk-1acts in a linear pathway by interacting with other upstream members ofthe insulin-like pathway.

Example 12 Phosphorylation of JNK-1 is Required for Life Span Extension

Dual phosphorylation of JNK on conserved Thr and Tyr by an upstreamkinase is critical for its function (10). The next question addressedwas whether the phosphorylation of JNK is required for life spanregulation. jnk-1 transgenic worms were crossed with loss-of-functionmutants of upstream kinases jkk-1(km2) and mek-1(ks54), and thephosphorylation status of JNK-1 was analyzed using a phospho-specificantibody that binds activated JNK. More phosphorylated JNK-1 wasdetected in the jnk-1 overexpression strain (lpIn2; FIG. 18). However,it was not detectable in either jnk-1(gk7) or jkk-1(km2) mutants. Inaddition, mutation in jkk-1 completely suppressed the phosphorylation ofJNK-1 in jnk-1 overexpression worms (lpIn2; FIG. 18). In accordance withthis result, life span extension by jnk-1 overexpression was alsosuppressed by mutation in jkk-1 (lpIn2: 18.8±0.5 days, jkk-1;lpIn2:14.9±0.4 days, N2+pRF4 control: 15.2±0.3 days; FIG. 19). However,mutation in mek-1(ks54), alone or in combination with jnk-1overexpression (lpIn2), did not affect JNK-1 phosphorylation (FIG. 18).These results show that JKK-1 is the upstream kinase of JNK-1 andphosphorylation of JNK-1 is required for life span extension.

Example 13 JNK-1 Interacts with and Phosphorylates DAF-16

Based on the genetic studies described above, it was next examinedwhether JNK-1 interacts physically with DAF-16. COS-7 cells weretransfected with plasmids encoding either Flag-tagged DAF-16 alone or incombination with Xpress-tagged JNK-1. Following coimmunoprecipitationwith anti-Xpress antibody, JNK-1 was found to bind to DAF-16 (FIG. 20A).It was then examined if DAF-16 could serve as a substrate for JNK-1.COS-7 cells were transfected with plasmids encoding Xpress-tagged JNK-1that was then immunoprecipitated from the cell lysate with anti-Xpressantibody and incubated with bacterially expressed N- or C-terminalportion of DAF-16 as a substrate in an in vitro kinase assay. Uponactivation of JNK-1 by UV, JNK-1 phosphorylates DAF-16 as well as themammalian c-Jun protein used as a positive control (FIG. 20B). Thephosphorylation was only observed with the N-terminal (83-307) fragmentof DAF-16, but not with the C-terminal (255-470) fragment (data notshown), implying that the JNK-1 phosphorylation site resides in theN-terminal region of DAF-16. Previously, JNK-1 was shown to be activatedby dual phosphorylation on Thr276 and Tyr278 (17, 19). Therefore, akinase-dead JNK-1 construct was created to determine specificity byreplacing TPY residues with APF, and the in vitro kinase assay wasperformed using the N-terminal portion of DAF-16 as a substrate. Thekinase-dead JNK-1 (APF) failed to phosphorylate DAF-16. These resultsconfirm the specificity of JNK-1 phosphorylation of DAF-16 as asubstrate (FIG. 21). These results also demonstrate that JNK-1 directlyinteracts with and phosphorylates DAF-16 as a separate input fromAKT-1/2. These results therefore support the parallel pathway modelsuggested by the genetic data described herein.

Example 14 Stress Resistance Conferred by Overexpression of jnk-1 isDependent on daf-16

JNK is a stress responsive gene in diverse organisms (10, 11).Accordingly, it was next examined whether overexpression of jnk-1confers stress resistance. Survival of young adult worms was monitoredat 35° C. jnk-1 transgenic lines showed significantly increasedresistance to heat stress (mean survival time N2+pRF4 control: 10.8±0.2hours, lpIn1: 15.3±0.3 hours, lpIn2: 14.4±0.2 hours (P<0.0001); FIG.22A). In addition, jnk-1 transgenic lines significantly increaseresistance to oxidative stress (FIG. 23).

In C. elegans, mutation in daf-2 or age-1 confers stress resistance andthis resistance is dependent on daf-16 (27-29). Accordingly, experimentswere carried out to determine if the stress resistance associated withjnk-1 overexpression strains was dependent on daf-16. The results ofthese experiments indicate that the stress resistance observed in thejnk-1 overexpression strains were also dependent on daf-16 (FIG. 24).

Example 15 Overexpression of jnk-1 Modulates Translocation of DAF-16 inResponse to Stress

In response to stress, DAF-16 translocates from the cytoplasm to thenucleus (30). Accordingly, experiments were carried out in which nucleartranslocation of DAF-16 was visualized in vivo in worms that overexpressjnk-1. To accomplish this, a jnk-1 overexpression line was crossed to astrain containing a daf-16::gfp reporter construct. A comparison wasmade between daf-16::gfp alone and lpIn2;daf-16::gfp orjkk-1;lpIn2;daf-16::gfp following 30 minutes of heat shock. The extentof nuclear translocation was categorized as Cyt (cytosolic), Nuc(nuclear) or Int (intermediate) (FIG. 22B) (28). The number of wormswith nuclear localization signal (Nuc) was increased in the jnk-1overexpression (lpIn2;daf-16::gfp) strain whereas that with cytosoliclocalization (Cyt) was decreased compared to the control (daf-16::gfp).However, mutation of jkk-1 suppressed the enhancement in nuclearlocalization in jnk-1 overexpression worms (daf-16::gfp (Cyt):16.7±1.4%, lpIn2;daf-16::gfp (Cyt): 4.3±0.9%: jkk-1;lpIn2;daf-16::gfp(Cyt): 21.0±2.4%, daf-16::gfp (Nuc): 13.6±1.8%, lpIn2;daf-16::gfp (Nuc):30.4±2.1%: jkk-1;lpIn2;daf-16::gfp (Nuc): 19.4±2.5%; FIG. 22B). Togetherwith the biochemical evidence described herein, this genetic analysissuggests that JNK-1 interacts directly with DAF-16 and modulates itsnuclear translocation in response to stress. Previous studies have alsoshown that daf-18, a phosphatase in the insulin-like signalling pathway,is also required for daf-16 translocation in response to stress (31).The data described herein would suggest that this is a novel input intodaf-16 for regulation of life span and stress.

Example 16 Identification of JNK Phosphorylation Sites on DAF-16

The specific sites of phosphorylation on DAF-16 by JNK-1 are identifiedby first identifying JNK MAPK consensus sequences in DAF-16. JNK MAPKphosphorylates Threonine or Serine residues followed by a Prolineresidue. DAF-16 has three isoforms, a1 and a2, which are similar, and bwhich differs in the N-terminus. Inspection of the DAF-16 sequencerevealed 8 putative JNK phosphorylation sites. By using N-terminal andC-terminal portions of DAF-16, the 3 putative JNK phosphorylation siteswere exclude, since the C-terminus failed to be phosphorylated. Theremaining putative JNK phosphorylation sites include T34 (Threonine),S69 (Serine), T107 (Threonine), S127 (Serine) and S160 (Serine). Inorder to identify the specific JNK phosphorylation site(s) in DAF-16,point mutations are introduced into DAF-16 or a portion thereof, e.g.,N-terminus, at each of the putative phosphorylation sites, whereThreonine or Serine is replaced with Alanine. The mutated substrate isthen purified and tested in a JNK kinase assay to determine if JNK canphosphorylate the individual mutants. A mutant that fails to bephosphorylated indicates that the JNK phosphorylation site is at theposition where the point mutation was introduced.

The identification of JNK phosphorylation sites in mammalian orthologuesof DAF-16, e.g., DAF-16/FOXO, can be identified in a similar manner.Additional methods of identifying the JNK phosphorylation sites inDAF-16 include testing DAF-16 mutants containing point mutations atputative phosphorylation sites in a DAF-16 translocation assay in cells,as described above in Examples 7 and 13. DAF-16 mutants which do nottranslocate to the nucleus upon exposure to stress would indicate thatthe phosphorylation site for JNK is at the position where the pointmutation was introduced.

Summary of Examples 6-16

Taken together, the results of the present invention suggest that theJNK signaling pathway can regulate lifespan by modulating nucleartranslocation of DAF-16 (model as depicted in FIG. 13). In particular,the results of the present invention suggest that the JNK signalingpathway is important to maintain normal lifespan and can extend lifespanwhen it is activated. This extension of lifespan appears to be mediatedby both the jkk-1 and mek-1 upstream kinases. Moreover, the resultspresented herein indicate that when the JNK signaling pathway isactivated, jnk-1 localizes DAF-16 into the nucleus where it enhances theexpression of stress resistant genes, thereby conferring stressresistance and extending life span.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

In addition, the contents of all patent publications discussed supra areincorporated in their entirety by this reference.

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1. A method for identifying an agent capable of modulating the physical interaction between c-jun N-terminal kinase 1 (JNK-1) or a mammalian ortholog thereof and DAF-16 or a mammalian ortholog thereof, comprising: contacting a cell with a test agent, said cell expressing JNK-1 or a mammalian ortholog thereof and DAF-16 or a mammalian ortholog thereof; and detecting physical interaction between said JNK-1 or mammalian ortholog thereof and said DAF-16 or mammalian ortholog thereof by examining the N-terminal phosphorylation and nuclear localization of said DAF-16 or mammalian ortholog thereof in the presence of said test agent as compared to a control cell to which no agent has been added; wherein an increase in the N-terminal phosphorylation and nuclear localization of said DAF-16 or mammalian ortholog thereof in the presence of said test agent identifies said agent as increasing the physical interaction between said DAF-16 or mammalian ortholog thereof and said JNK-1 or mammalian ortholog thereof, and wherein a decrease in the N-terminal phosphorylation and nuclear localization of said DAF-16 or mammalian ortholog thereof in the presence of said test agent identifies said agent as decreasing the physical interaction between said DAF-16 or mammalian ortholog thereof and said JNK-1 or mammalian ortholog thereof.
 2. The method of claim 1, wherein the cell is a mammalian cell.
 3. The method of claim 1, wherein the cell is a human cell.
 4. The method of claim 1, wherein the cell is a bacterial cell or a yeast cell.
 5. The method claim 1, wherein the cell is a cell derived from a nematode.
 6. A method for identifying an agent capable of modulating the physical interaction between JNK-1 or a mammalian ortholog thereof and DAF-16 or a mammalian ortholog thereof, comprising: contacting an assay composition with a test compound, wherein said assay composition comprises JNK-1 or a mammalian ortholog thereof and DAF-16 or a mammalian ortholog thereof; and detecting the phosphorylation of the N-terminal region of said DAF-16 or mammalian ortholog thereof as compared to a control assay composition to which no agent has been added; wherein an increase in the phosphorylation of the N-terminal region of said DAF-16 or mammalian ortholog thereof in the presence of said agent identifies said agent as increasing the physical interaction between said JNK-1 or mammalian ortholog thereof and said DAF-16 or mammalian ortholog thereof, and wherein a decrease in the phosphorylation of the N-terminal region of said DAF-16 or mammalian ortholog thereof in the presence of said agent identifies said agent as decreasing the physical interaction between said JNK-1 or mammalian ortholog thereof and said DAF-16 or mammalian ortholog thereof.
 7. The method of claim 6, wherein said assay composition is a cell-free extract.
 8. The method of claim 6, wherein said assay composition comprises purified proteins.
 9. The method of claim 1 or 6, wherein said agent is identified based on its ability to decrease said physical interaction.
 10. The method of claim 1 or 6, wherein said agent is identified based on its ability to increase said physical interaction.
 11. The method of claim 1 or 6, wherein said agent is identified based on its ability to increase the N-terminal phosphorylation of said DAF-16 or mammalian orthologue thereof.
 12. A method of identifying an agent that modulates the phosphorylation of DAF-16 or a mammalian ortholog thereof by c-jun N-terminal kinase 1 (JNK-1) or a mammalian ortholog thereof comprising: contacting a cell with a test agent, said cell expressing JNK-1 or a mammalian ortholog thereof and DAF-16 or a mammalian ortholog thereof; detecting phosphorylation of the N-terminal region of said DAF-16 or mammalian ortholog thereof in the presence of said test agent as compared to a control cell to which no agent has been added; wherein an increase in said phosphorylation of said N-terminal region of said DAF-16 or mammalian ortholog thereof in the presence of said test agent identifies said agent as increasing the phosphorylation of said DAF-16 or mammalian ortholog thereof by said JNK-1 or mammalian ortholog thereof, and wherein a decrease in said phosphorylation of said N-terminal region of said DAF-16 or mammalian ortholog thereof in the presence of said test agent identifies said agent as decreasing the phosphorylation of DAF-16 or mammalian ortholog thereof by said JNK-1 or mammalian ortholog thereof.
 13. The method of claim 12, wherein the cell is a mammalian cell.
 14. The method of claim 12, wherein the cell is a human cell.
 15. The method of claim 12, wherein the cell is a bacterial cell or a yeast cell.
 16. The method of claim 12, wherein the cell is a cell derived from a nematode.
 17. A method for identifying an agent capable of modulating the physical interaction between c-jun N-terminal kinase 1 (JNK-1) or a mammalian ortholog thereof and DAF-16 or a mammalian ortholog thereof, comprising: contacting an assay composition with a test agent, wherein said assay composition comprises JNK-1 or a mammalian ortholog thereof and DAF-16 or a mammalian ortholog thereof; and examining the co-immunoprecipitation of said JNK-1 or mammalian ortholog thereof and said DAF-16 or mammalian ortholog thereof in the presence of said test agent as compared to a control assay composition to which no agent has been added; wherein an increase of said co-immunoprecipitation in the presence of said test agent identifies said agent as increasing the physical interaction between said DAF-16 or mammalian ortholog thereof and said JNK-1 or mammalian ortholog thereof, and wherein a decrease of said co-immunoprecipitation in the presence of said test agent identifies said agent as decreasing the physical interaction between said DAF-16 or mammalian ortholog thereof and said JNK-1 or mammalian ortholog thereof. 